Novel methods

ABSTRACT

The present invention relates to the field of methods for providing components of pharmaceutical compositions comprising poorly water-soluble drugs. In particular the present invention relates to methods for providing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, useful in pharmaceutical compositions.

FIELD OF THE INVENTION

The present invention relates to the field of methods for providing components of pharmaceutical compositions comprising poorly water-soluble drugs. In particular the present invention relates to methods for providing hybrid nanoparticles of protein kinase inhibitors (PKIs), in order to increase the dissolution rate and resulting bioavailability of said PKIs, useful in pharmaceutical compositions.

BACKGROUND OF THE AND INVENTION

Components of cellular signal transduction pathways that regulate the growth and differentiation of normal cells can, when dysregulated, lead to the development of cellular proliferative disorders and cancer. Mutations in cellular signaling proteins may cause such proteins to become expressed or activated at inappropriate levels or at inappropriate times during the cell cycle, which in turn may lead to uncontrolled cellular growth or changes in cell-cell attachment properties.

Many proliferative disorders, such as tumors and cancers, have been shown to involve overexpression or upregulation of protein kinase activity. Protein kinases are kinase enzymes that modify proteins by chemically adding phosphate groups (phosphorylation). Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins. Protein kinases can be subdivided or characterised by the amino acids of the target protein whose phosphorylation they control: most kinases act on both serine and threonine, the tyrosine kinases act on tyrosine, and a number (dual-specificity kinases) act on all three. There are also protein kinases that phosphorylate other amino acids, including histidine kinases that phosphorylate histidine residues. The human genome contains about 500 protein kinase genes and up to 30% of all human proteins may be modified by protein kinases. Kinases are known to regulate the majority of cellular pathways, especially those involved in signal transduction. Dysregulation of protein kinases by mutation, gene rearrangement, gene amplification, and overexpression of both receptor and ligand has been implicated in the development and progression of human cancers. Protein kinase inhibiting compounds or protein kinase inhibitors (PKIs) are therefore useful for treating diseases caused by or exacerbated by overexpression or upregulation of protein kinases. For example, tyrosine kinase inhibitors (TKIs also known as tyrphostins) have been shown be effective anti-tumor agents and anti-leukemic agents (Lowery A et. al., Front Biosci. 2011 Jun. 1; 17:1996-2007).

A major objective of formulation chemistry is to improve drug efficiency and safety, by e.g. improving bioavailability and stability as well as convenience to the patient. Bioavailability means the rate and extent to which an active substance or therapeutic is absorbed from a pharmaceutical form and becomes available at the site of action. The most common and preferred method of delivery due to convenience, ease of ingestion, and high patient compliance to treatment is the oral route of drug delivery. However, for certain drugs, drug absorption from the gastrointestinal tract is limited by poor aqueous solubility and/or poor membrane permeability of the drug molecules.

PKIs are generally weak bases that dissolve only slightly at low pH (e.g. 100-1000 mg/L) and are practically insoluble at neutral pH (e.g. 0.1-10 mg/L). Therefore, enhancing the solubility and dissolution rate of PKI-based drugs is important for improving the bioavailability and efficacy of most of these drugs. Typical PKIs exhibit non-polypeptide structure and have relatively low molecular weights, such as 10000 dalton or 5000 dalton.

Several methods to improve the dissolution characteristics of poorly water soluble drugs have been reported, including micronisation, formation of salts or solvates, complexes and microspheres. Additionally, attempts have been made to improve bioavailability provided by solid dosage forms by forming particles comprising the drug or by mixing the poorly water soluble drug with hydrophilic excipients. Traditionally, however, these methods carry inherent limitations concerning physical stabilities of the particles on storage, problems with grinding or difficulty of removal of the frequently toxic solvent. Furthermore, it is important that the drug released from the solid phase does not precitipitate in the gastrointestinal tract, or precipitates as little as possible, but remains water-soluble in the aqueous fluids of the gastrointestinal tract, since such precipitation results in low bioavailability (see e.g. Hervé J. et al. Pharm Dev Technol. 2011 June; 16(3):278-86).

pH-dependent solubility is a well-known issue for many oral formulations of poorly water-soluble substances, such as PKIs, since most of the absorption of the drug occurs in the small and large intestine, where pH is close to neutral. There is thus a continuing need to develop and improve the dissolution characteristics of oral solid dosage forms of PKI-based drugs. (Budha N R, Frymoyer A, Smelick G S, Jin J Y, Yago M R, Dresser M J, Holden S N, Benet L Z, Ware J A. Clin Pharmacol Ther. 2012 August; 92(2):203-13). Therefore, methods for improving dissolution of PKI-based drugs, as well as of other poorly water-soluble drugs, at neutral (intestinal) pH are highly desirable.

US20090203709 discloses a pharmaceutical dosage form comprising a solid dispersion product of at least one tyrosine kinase inhibitor, at least one pharmaceutically acceptable polymer and at least one pharmaceutically acceptable solubilizer. Further the reference discloses methods for preparing the above-mentioned pharmaceutical dosage form, comprising preparing the homogenous melt of at least one tyrosine kinase inhibitor, at least one pharmaceutically acceptable polymer and at least one pharmaceutically acceptable solubilizer, and allowing the melt to solidify to obtain a solid dispersion product.

EP2105130 discloses pharmaceutical formulations comprising a solid dispersion or solid solution, containing a polymer and an active agent in amorphous form. Further, the formulation comprises an external polymer to stabilize the solution, such that the % by weight of the external polymer is less than 20% of the total weight of the pharmaceutical formulation. Additionally, the reference discloses a hot melt extrusion method for production of the above-mentioned formulation.

SUMMARY OF THE INVENTION

The present invention relates to methods of producing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component. Optionally, one or more solubilizers may be added to the particles, present separately from the particles, or within the particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising nilotinib HCl produced by the methods of the invention. Further experimentation with both nilotinib base and nilotinib HCl is found in Example 1. The details of the particles are described in Example 1, Table 1, for experiment 3, 30 and 37, respectively. Briefly, experiment 30 represents hybrid nanoparticles comprising nilotinib HCl and HPMCP HP55 and wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. Experiment 3 represents raw, crystalline nilotinib HCl and experiment 37 represents hybrid nanoparticles of nilotinib HCl, HPMCP HP55 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, present within the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5, in FaSSIF.

FIG. 2 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising erlotinib produced by the methods of the invention. Further experimentation with erlotinib is found in Example 2. The details of the hybrid nanoparticles are described in Example 2, Table 7, for experiment 58, 65 and 67, respectively. Briefly, experiment 65 represents hybrid nanoparticles with erlotinib HCl and HPMC-AS, wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. Experiment 58 represents raw, crystalline erlotinib HCl and experiment 67 represents hybrid nanoparticles of erlotinib HCl, HPMC-AS and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer present within the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 3 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising pazopanib produced by the methods of the invention. Further experimentation with pazopanib is found in Example 3. The details of the hybrid nanoparticles are described in Example 3, Table 13, for experiment 84, 91 and 93, respectively. Briefly, experiment 91 represents hybrid nanoparticles comprising pazopanib and PVP 90K and wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles, experiment 93 represents hybrid nanoparticles comprising pazopanib, PVP 90K and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, present within the hybrid nanoparticles. Experiment 84 represents raw, crystalline pazopanib. The experiments illustrated in the graphs were carried out at pH 6.5, in FaSSIF.

FIG. 4 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising lapatinib base produced by the methods of the invention. Further experimentation with both lapatinib base and lapatinib ditosylate salt is found in Example 4. The details of the hybrid nanoparticles are described in Example 4, Table 19, for experiment 110, 122 and 126, respectively. Briefly, experiment 122 represents hybrid nanoparticles comprising lapatinib base and HPC EF, wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. Experiment 110 represents raw, lapatinib base and experiment 126 represents hybrid nanoparticles of lapatinib base, HPC LF and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer present within the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 5 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles particles comprising nilotinib produced by the methods of the invention. The details of the hybrid nanoparticles are described in Example 5, Table 21, for experiment 127, 128 and 129, respectively. Briefly, experiment 129 represents a physical mixture of raw, crystalline nilotinib HCl, HPMCP HP55 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 128 represents hybrid nanoparticles comprising nilotinib HCl and HPMCP HP55, wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. Experiment 127 represents hybrid nanoparticles of nilotinib HCl and HPMCP HP55. The experiments illustrated in the graphs were carried out at pH 1.4 in SGF.

FIG. 6 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising gefitinib produced by the methods of the invention. Further experimentation with gefitinib is found in Example 6 The details of the compositions are described in Example 6, Table 22, for experiment 131, 133, 135 and 137, respectively. Briefly, experiment 131 represents raw, crystalline gefitinib. Experiment 133 represents a mixture of raw, crystalline gefitinib, HPMCP HP55 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 135 represents hybrid nanoparticles of gefitinib and HPMCP HP55. Experiment 137 represents hybrid nanoparticles of gefitinib and HPMCP HP55 wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 7 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising dasatinib produced by the methods of the invention. The details of the hybrid nanoparticles are described in Example 7, Table 24, for experiments 138-141. Briefly, experiment 138 represents raw, crystalline dasatinib. Experiment 139 represents a mixture of raw, crystalline dasatinib, Kollidon VA64 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 140 represents hybrid nanoparticles of dasatinib and Kollidon VA64. Experiment 141 represents hybrid nanoparticles of dasatinib and Kollidon VA64 wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 8 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising sorafenib produced by the methods of the invention. The details of the hybrid nanoparticles are described in Example 8, Table 26, for experiments 142-145. Briefly, experiment 142 represents raw, crystalline sorafenib tosylate. Experiment 143 represents a mixture of raw, crystalline sorafenib tosylate, HPMCP HP55 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 144 represents hybrid nanoparticles of sorafenib tosylate and HPMCP HP55. Experiment 145 represents hybrid nanoparticles of sorafenib tosylate and HPMCP HP55 wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 9 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising crizotinib produced by the methods of the invention. Further experimentation with crizotinib is found in Example 10. The details of the compositions are described in Example 10, Table 30, for experiment 150, 152, 153 and 156, respectively. Briefly, experiment 150 represents raw, crystalline crizotinib. Experiment 152 represents a mixture of raw, crystalline crizotinib, PVP 30K and the solubilizer Cremophor RH40. Experiment 153 represents hybrid nanoparticles of crizotinib and PVP 30K. Experiment 156 represents hybrid nanoparticles of crizotinib and PVP 30K wherein the solubilizer Cremophor RH40 is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 10 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising axitinib produced by the methods of the invention. Further experimentation with axitinib is found in Example 11 The details of the compositions are described in Example 11, Table 32, for experiment 157, 158, 160 and 162, respectively. Briefly, experiment 157 represents raw, crystalline axitinib. Experiment 158 represents a mixture of raw, crystalline axitinib, Kollidon VA64 and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 160 represents hybrid nanoparticles of axitinib and Kollidon VA64. Experiment 162 represents hybrid nanoparticles of axitinib and Kollidon VA64 wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 11 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles comprising vemurafenib produced by the methods of the invention. Further experimentation with vemurafenib is found in Example 12. The details of the compositions are described in Example 12, Table 34, for experiment 164, 166, 168 and 170, respectively. Briefly, experiment 164 represents raw, crystalline vemurafenib. Experiment 166 represents a mixture of raw, crystalline vemurafenib, CAP and the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Experiment 168 represents hybrid nanoparticles of vemurafenib and CAP. Experiment 170 represents hybrid nanoparticles of vemurafenib and CAP wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer is present separately from the hybrid nanoparticles. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 12 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising nilotinib base produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.1, and Table 36 for experiments 500 and 501. Briefly, experiment 500 represents raw, nilotinib HCl. Experiment 501 represents hybrid nanoparticles of nilotinib base and HPMCP HP55. The experiments illustrated in the graphs were carried out at pH 6.5 in FaSSIF.

FIG. 13 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising erlotinib HCl produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.2, Table 37 for experiments 510 and 511. Briefly, experiment 510 represents raw, erlotinib HCl. Experiment 511 represents hybrid nanoparticles of erlotinib HCl and HPMC AS.

FIG. 14 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising pazopanib HCl produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.3, Table 38 for experiments 520 and 521. Briefly, experiment 520 represents raw, pazopanib HCl. Experiment 521 represents hybrid nanoparticles of pazopanib HCl and PVP90K.

FIG. 15 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising lapatinib base produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.4, Table 39 for experiments 530 and 531. Briefly, experiment 530 represents raw, lapatinib ditosylate. Experiment 531 represents hybrid nanoparticles of lapatinib base and HPC If.

FIG. 16 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising gefitinib produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.5., Table 40 for experiments 540 and 541. Briefly, experiment 540 represents raw, gefitinib. Experiment 541 represents hybrid nanoparticles of gefitinib and HPMCP HP55.

FIG. 17 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising dasatinib produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.6., Table 41 for experiments 550 and 551. Briefly, experiment 550 represents raw, dasatinib. Experiment 551 represents hybrid nanoparticles of dasatinib and Kollidon VA64.

FIG. 18 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising sorafenib tosylate produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.7., Table 42 for experiments 560 and 561. Briefly, experiment 560 represents raw, sorafenib tosylate. Experiment 561 represents hybrid nanoparticles of sorafenib tosylate and HPMCP HP55.

FIG. 19 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising crizotinib produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.8., Table 43 for experiments 570 and 571. Briefly, experiment 570 represents raw, crizotinib. Experiment 571 represents hybrid nanoparticles of crizotinib and PVP 30K.

FIG. 20 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising axitinib produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13. 9., Table 44 for experiments 580, 581 and 582. Briefly, experiment 580 represents raw, axitinib. Experiment 581 represents hybrid nanoparticles of axitinib and Kollidon VA64 and experiment 582 represents hybrid nanoparticles of axitinib and HPMC AS.

FIG. 21 provides a graph showing the dissolution rate for stable, amorphous hybrid nanoparticles comprising vemurafenib produced by the methods of the invention, measured under sink conditions. Details are found in Examples 13 and 13.10., Table 45 for experiments 590, 591 and 592. Briefly, experiment 590 represents raw, vemurafenib. Experiment 591 represents hybrid nanoparticles of vemurafenib and Kollidon VA64 and experiment 592 represents hybrid nanoparticles of vemurafenib and CAP.

FIG. 22 provides graphs showing in vivo measurement of plasma levels after oral administration to beagle dogs of compositions, represented by formulations comprising stable, amorphous hybrid nanoparticles of nilotinib base and the polymeric stabilizing and matrix-forming components PVAP and HPMCP HP55, respectively (I/P), denoted PVAP and HP55, as well as wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added (I/P+S), denoted HP55s and PVAPs, respectively. The experiments were carried out in beagle dogs pre-treated to have neutral stomach content. The hybrid nanoparticles are further described in experiments 146 and 147 (Example 9) and details of the in vivo experiments are set out in Example 14. The experiments used a marketed formulation comprising nilotinib HCl (“Tasigna”) as reference.

FIG. 23 provides graphs showing in vivo measurement of plasma levels after oral administration to beagle dogs of compositions, represented by formulations comprising stable, amorphous hybrid nanoparticles of nilotinib base and the polymeric stabilizing and matrix-forming components PVAP and HPMCP HP55, respectively (I/P), denoted PVAP and HP55, produced by the methods of the invention as well as wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added (I/P+S), denoted PVAPs and HP55s, respectively. The experiments were carried out in beagle dogs pre-treated to have acidic stomach content. The hybrid nanoparticles are further described in experiments 146 and 147 (Example 9) and details of the in vivo experiments are set out in Example 14. The experiments used a marketed formulation comprising nilotinib HCl (“Tasigna”) as reference.

FIG. 24 provides graphs showing in vivo measurement of plasma levels after oral administration to beagle dogs of compositions, represented by formulations comprising stable, amorphous hybrid nanoparticles of nilotinib base and the polymeric stabilizing and matrix-forming components PVAP and HPMCP HP55, respectively (I/P) produced by the methods of the invention, denoted PVAP and HP55, as well as wherein the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added after hybrid nanoparticle formation (I/P+S), denoted PVAPs and HP55s, respectively. The experiments were carried out in beagle dogs pre-treated to have acidic or neutral stomach content. The hybrid nanoparticles are further described in experiments 146 and 147 (Example 9) and details of the in vivo experiments are set out in Example 14.

FIG. 25 provides graphs showing in vivo measurement of plasma levels after oral administration to beagle dogs of compositions, represented by formulations comprising stable, amorphous hybrid nanoparticles of nilotinib base and the polymeric stabilizing and matrix-forming components PVAP and HPMCP HP55, respectively (I/P) produced by the methods of the invention, denoted PVAP and HP55. The experiments were carried out in beagle dogs pre-treated to have acidic or neutral stomach content. The hybrid nanoparticles are further described in experiments 146 and 147 (Example 9) and details of the in vivo experiments are set out in Example 14.

FIG. 26 provides a graph showing the apparent solubility for stable, amorphous hybrid nanoparticles produced by the methods of the invention, before and after 11 months of storage at room temperature. The experiment provides stable, amorphous hybrid nanoparticles comprising nilotinib base, HPMCP HP55 and the addition of the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (I/P+S) as Exp 171 & Exp 172 with further details set out in Example 15.

FIG. 27 provides overlayed X-ray powder diffraction (XRPD) patterns of stable hybrid nanoparticles at 40% drug load, I/P nilotinib base/HPMCP HP55. Initial (top) and after 12 months storage at ambient temperature (bottom). The XRPD patterns are offset in order improve the visual comparison. Further details are set out in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

As used herein, the phrase “hybrid nanoparticles” refers to a group of particles, typically in the average size range of from 1 to 1000 nm, composed of at least two components, one of which is the PKI and the other a polymeric stabilizing and matrix-forming component. The particles can be either crystalline or amorphous, or a mixture thereof. Typically, in the sense of the present disclosure, the particles are “amorphous”, or “essentially amorphous”. This means that almost all, if not all, content of the particles comprise amorphous protein kinase inhibitor and polymeric stabilizing and matrix-forming component. The level or degree of amorphicity is at least 60%, such as 70%, such as 80% or 85%, preferably at least 90% and more preferably >95%, wherein 100% represents that all material is amorphous in the particles. Quantification of crystalline PKI or absence of crystalline PKI may be measured by X-ray powder diffraction methods as described in Saleki-Gerhardt A et al. Int J Pharm. 1994; 101:237-247) or by water vapor sorption as described in Dash A K et al. J Pharm Sci. 2002 April; 91(4):983-90.

The term “solid dispersion particles” relates to “hybrid nanoparticles” as defined above, however, solid dispersion particles are typically larger or much larger in size (typically μm-mm, as described in Wu K. et al. J Pharm Sci. 2009 July; 98(7):2422-3). The smaller size of hybrid nanoparticles contributes to further stabilizing the PKI against crystallization. Typically, hybrid nanoparticles is in the average size range of from 1 to 1000 nm, such as below 500 nm, preferably below 250 nm.

The phrase “stable” refers to the level of stability of produced particles by the methods of the present invention and may be measured as the capability of the hybrid nanoparticles to remain in their physical state for 6-12 months storage at ambient temperature (e.g. 20-25° C.). The level of stability may be measured by AUC measurements of dissolution rate over for instance 80 minutes of the particles, after such storage.

By the phrase “protein kinase inhibitor” or “PKI” is meant a type of enzyme inhibitor that specifically blocks the action of one or more protein kinases. PKIs include, but are not limited, to protein kinase inhibitors and tyrosine kinase inhibitors, such as axitinib, afatinib, bosutinib, crizotinib, cediranib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, lestaurtinib, motesanib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, semaxanib, sunitinib, tandunitib, tipifamib, vandetanib and vemurafenib; or salts or hydrates or solvates thereof, or combinations thereof.

By the phrase “polymeric stabilizing and matrix-forming component” is meant the component present in the hybrid nanoparticles together with the PKI. Typically, said polymeric stabilizing and matrix-forming component exhibits a polymeric structure, such as, but not limited to, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. HPC ef, HPC If and HPC jf), hydroxypropyl methylcellulose (e.g. Methocel E3 and E15 and Pharmacoat), hydroxypropyl methylcellulose acetate succinate (HPMC AS), hydroxypropyl methylcellulose phthalate (e.g. HPMCP-HP55), polyvinylpyrrolidone (e.g. PVP 30K and PVP 90K), polyvinyl acetate phthalate (PVAP), copolyvidone (e.g. Kollidon VA 64), crospovidon (e.g. Kollidon CL), methacrylic acid and ethylacrylate copolymer (e.g. Kollicoat ME), methacrylate acid and methyl methacrylate copolymer (e.g. Eudragit L100), polyethylene glycol (PEG), DL lactide/glycolide copolymer, poly DL-lactide, cellulose acetate phthalate (CAP), aminoalkyl methacrylate copolymers (e.g. Eudragit RL100, RL PO or RS PO), carbomer homopolymer Type A (e.g. Carbopol 971P), carbomer homopolymer Type B (e.g. Carbopol 974P) and Poloxamers (e.g. Pluronics, Kolliphor).

The term “polymer” or “polymeric” is here used to mean a compound that is made of monomers connected together to form a larger molecule. A polymer generally consists of 20 or more monomers connected together, however less than 20 monomers connected together are here also referred to as polymers.

The term “solubilizer” is here used to mean a compound that increases the solubility of a substance, such as, but not limited to, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus), d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS), PEG-40 hydrogenated castor oil (Cremophor RH40), PEG-35 castor oil (Cremophor EL), PEG-40 stearate (MYRJ 540), hard fat (e.g. Gelucire 33/01), polyoxylglycerides (e.g. Gelucire 44/14), stearoyl polyoxylglycerides (e.g. Gelucire 50/13), PEG-8 caprylic/capric glycerides (e.g. Labrasol) and Poloxamers (e.g. Pluronics, Kolliphor).

As used herein, the phrase “primary particles” refers to the smallest particulate entities formed during the precipitation process. The boundaries of the particles are analyzed by SEM microscopy. Depending on process parameters, the primary particles may build together a more or less dense and porous network forming larger, agglomerated or bridging particles. Parameters affecting the agglomeration are e.g. temperature that may modify the softness of the primary particles; ratio solvent/antisolvent affecting precipitation time, concentration of the PKI solution; and the nature of the polymeric stabilizing and matrix-forming agent(s). The average size of the primary particles is typically between 1 to 1000 nm, preferably below 500 nm, more preferably below 250 nm.

As used herein, the phrases “supercritical” and “supercritical fluid” refer to that a chemical substance that is set to both a temperature higher or equal than its critical temperature (Tc) and a pressure higher or equal than its critical pressure (Pc).

As used herein, the phrases “subcritical” and “subcritical fluid” refer here to that one of critical temperature (Tc) or critical pressure (Pc) is set to a temperature or pressure higher than its critical temperature (Tc) or critical pressure (Pc), respectively, and the other of critical temperature (Tc) or critical pressure (Pc) is set to a temperature or pressure lower than its critical temperature (Tc) or critical pressure (Pc), respectively.

By the phrase “area under the curve (AUC)” is meant the area under the concentration-time curve, where the x-axis represents time and the y-axis represents solubilized drug concentration.

By the phrase “apparent solubility” is meant the concentration of material at apparent equilibrium. See further in the Examples section.

The term “supersaturation” is here used to mean that a solution contains more of the dissolved substance than could be dissolved by the solvent or media under normal circumstances.

As used herein, the term “Soluplus” or “soluplus” refers to polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer.

As used herein, the term “TPGS” refers to d-α-tocopherol acid polyethylene glycol 1000 succinate.

As used herein, the term “Chremophor RH40” refers to PEG-40 hydrogenated castor oil.

As used herein, the term “PVAP” refers to polyvinyl acetate phthalate.

As used herein, the term “PVP 90K” refers to polyvinylpyrrolidone K-90.

As used herein, the term “PVP 30K” refers to polyvinylpyrrolidone K-30.

As used herein, the term “HPMC-AS” refers to hydroxypropyl methylcellulose acetate succinate.

As used herein, the term “HPMCP HP55” refers to hydroxypropyl methyl cellulose phthalate.

As used herein, the term “HPC” refers to hydroxypropyl cellulose, such as HPC EF and HPC LF.

As used herein, the term “Kollidon VA64” refers to copolyvidone.

As used herein, the term “CAP” refers to cellulose acetate phthalate.

The dissolution mediums used for purposes of testing hybrid nanoparticles produced by the methods of the present invention, includes Fasted State Stimulated Intestinal Fluid, referred to as FaSSIF, Fed State Stimulated Intestinal Fluid, referred to as FeSSIF, and Simulated Gastric Fluid, referred to as SGF. FaSSIF media is tailored to represent a fasting state and has a pH of about 6.5 as well as particular osmolaric properties. FeSSIF media is tailored to represent a fed state and has a pH of about 5 as well as specific osmolaric properties. SGF is tailored to represent gastric fluid and has a pH of about 1.4 as well as particular osmolaric properties. FaSSIF, FeSSIF and SGF media are generally used in in vitro models for dissolution of poorly water-soluble drugs. The choice of medium will be dependent of the where in the intestinal tract and under what conditions (fasted or fed) particles are desired to dissolve and be taken up. Further details regarding these fluids are described in e.g. Hervé J. et al. Pharm Dev Technol. 2011 June; 16(3):278-86 and Jantratid, E., and Dressman, J. Dissolut. Technol. 2009 8, 21-25.

By the phrase “amorphous form” is meant non-crystalline solid form. The ease of dissolution may at least in part be attributed to the amount of energy required for dissolution of the components from a crystalline or amorphous solid phase. Amorphous particles require less energy for dissolution as compared to crystalline particles of the same compound.

The inventive methods produce hybrid nanoparticles comprising a PKI or a combination of two or more PKIs. However, the particles may comprise a combination of one or more PKIs and at least one further active ingredient, such as one or more drugs. Various kinds of PKIs can be effectively utilized.

The term PKIs (protein kinase inhibitors) as used herein, is intended to include also the hydrates, solvates (alcoholates) pharmaceutically acceptable acid salts, base salts or co-crystals of such protein kinase inhibiting compounds.

As used herein, the term water-insoluble or poorly water soluble (or hydrophobic) compounds, refers to compounds whose solubility in water at 25° C. is less than 1 g/100 ml, especially less than 0.1 g/100 ml in pure water at neutral pH.

The hybrid nanoparticles generated by the methods of the present invention are typically in the form of particles as described elsewhere in this specification. There are a number of different methods for the formation of larger particles, e.g. granulation, melt extrusion, spray drying, precipitation etc. all of which typically encompass starting with formation of a mixture between the Active Pharmaceutical Ingredient (API) and the polymeric stabilizing and matrix-forming component. The inventive methods of the present invention are continuos processes for generating hybrid nanoparticles. Continuous processes in this context means that particle formation is continuously ongoing while at the same time continuously withdrawing/collecting/retaining hybrid nanoparticles from the mixture after their formation. In the preferred methods, i.e. precipitation methods, this means that a fluid which is a solution of the PKI, preferably in the form of a fluid stream, is mixed with an antisolvent fluid, preferably in the form of an antisolvent fluid stream. The polymeric stabilizing and matrix-forming component may be present in either one or both of the two fluids depending on its solubility characteristics. The mixing of the two fluids is taking place in a mixing function, e.g. a mixing chamber. In the case the process is continuous, i.e. the two fluids are fluid streams, the mixing function typically is associated with a particle formation and separation function wherein the mixed fluid stream may pass through while retaining the hybrid nanoparticles. Agents modifying the particle characteristics without being incorporated into the particles may be added to either one or both of the two fluids before the mixing step. The fluids typically are conventional liquids or supercritical fluids, where supercritical fluids also include subcritical fluids (i.e. fluids for which only one of pressure and temperature is above its supercritical value). Typical combinations are, a) conventional (i.e., non-supercritical) liquids for both the API solution and the antisolvent, b) supercritical solution of the API combined with conventional liquid for the antisolvent, c) conventional liquid for the API solution combined with supercritical fluid for the antisolvent, and d) supercritical fluids for both of the two fluids. In certain variants the antisolvent may be omitted. A fluid stream, preferably supercritical, containing both the API and the polymeric stabilizing and matrix-forming component is then allowed to expand into the particle formation function. It is preferred that at least one of the fluids is in a supercritical state in the precipitation methods described above. These kinds of precipitation methods are discussed in WO 2005061090 (Censdelivery AB), WO 2009072950 (XSpray Microparticles AB), WO 2009072953 (XSpray Microparticles AB), WO 2011159218 (XSpray Microparticles AB) and references cited in these publications.

The term “solution” encompasses that the solute is either a true solute or minute particles of colloidial dimensions (typically 1-1000 nm) and less than the particles to be produced.

A preferred particle formation system is the “Right Size system” developed by XSpray Microparticles AB, Sweden. A detailed description of the technology can be found in the WO-publications given in the preceding paragraph. An important characteristic of the system is that the two fluid streams should merge within a nozzle at an angle in the interval 45°-135°, with preference for about 90° and sprayed into a particle formation/separation function. In principle the system allows for producing particles of predetermined size and/or morphology. Here the Right Size system and apparatus will be described using the non-limiting example of a PKI as the drug and CO₂ as a supercritical fluid antisolvent.

The system consists of one pumping set-up for the PKI dissolved in a liquid solvent, referred to as the API solution, and one pumping set-up for an antisolvent, for example CO₂, however also other antisolvents may be used when suitable. Each pumping set-up includes instruments such as a flow meter and a pressure meter that are used to control the process conditions. These two pumping set-ups are fluidically connected at a spray nozzle.

A stream of liquid API solution is mixed with a stream of CO₂ under flow conditions within the spray nozzle. The polymeric stabilizing and matrix-forming component is present in either the API solution or in the stream of CO₂. These streams are sprayed at the outlet of the nozzle into a precipitation vessel under controlled conditions (typically pressure and temperature). CO₂ acts as an antisolvent and makes the API to precipitate together with the polymeric stabilizing and matrix-forming component into fine particles. Particles are retained in the vessel by a filtering set-up. A back pressure regulator is typically used to control the pressure inside the precipitation vessel.

For preparing hybrid nanoparticles of certain drugs, for example but not limited to Pazopanib and Erlotinib, it may be advantageous to have an extra pumping set-up for injecting an additional solvent, referred to as a modifier, into the CO₂. Here a pumping set-up control is set up for the modifier and the modifier is mixed with the CO₂ in a mixer before entering the nozzle.

When using the system, the system operator typically starts by equilibrating the system by pumping CO₂, an “PKI like solution” (a solution similar in composition to the PKI solution but containing no PKI and no excipient) and the modifier (if used) through the system until flow rates, pressure and temperature have reached a desired steady state. Critical parameters for setting up the system are PKI solution composition, PKI solution flow rate, CO₂ flow rate, CO₂ pressure and temperature, nature of the modifier and modifier flow rate, if such is used.

Next, the “PKI like solution” is exchanged for the PKI solution and particles are produced and retained downstream of the mixing, e.g. downstream of the outlet of the nozzle. Afterwards, the system is typically cleaned by pumping the “PKI like solution” through the system. The particles are dried by flushing CO₂ through the retained particles in order to extract any remaining solvent. The precipitation vessel is then depressurized and the particles can be collected.

The solution/solvent and the antisolvent are typically miscible with each other. The pressure and temperature in the particle formation function, and/or upstream of this function, such as in the mixing function, provide supercritical or subcritical conditions in relation to the antisolvent.

The concentration of the PKI in the solution is typically below its saturation concentration, such as ≦50%, such as ≦60%, such as ≦75%, such as ≦85% or such as ≦95% of the saturation concentration. Suitable concentrations are typically found in the interval ≦20%, such as ≦10% or ≦5% or ≦3% with lower limits being ≦005% or 0.1% (all in w/v-%). The term “volatile” for solvents typically means boiling points of ≦200° C., such as ≦150° C. or ≦100° C., at atmospheric pressure. Examples are inorganic solvents and organic solvents with particular emphasis of dimethyl sulfoxide and trifluoroethanol and mixtures thereof. The term solvent includes mixtures of liquids which are miscible with each other. The solutions may contain agents that enhance or diminish the solubility of the PKI, e.g. acidic, alkaline, buffer components and/or other organic solvents.

Illustrative fluids which can be used as an antisolvent are

-   -   a) gaseous at room temperature and atmospheric pressures, or     -   b) liquid at room temperature and atmospheric pressure.

The antisolvent is typically selected for its ability to be readily dispersed into small droplets and for its ability to act as an atomizing agent and antisolvent against the PKI present in the solution.

Compounds/elements according to group (a) may be selected from carbon dioxide (Pc=74 bar and Tc=31° C.) (preferred), nitrous oxide (Pc=72 bar and Tc=36° C.), sulphur hexafluoride (Pc=37 bar and Tc=45° C.), ethane (Pc=48 bar and Tc=32° C.), ethylene (Pc=51 bar and Tc=10° C.), xenon (Pc=58 bar and Tc=16° C.), trifluoromethane (Pc=47 bar and Tc=26° C.), chlorotrifluoromethane (Pc=39 bar and Tc=29° C.) and nitrogen (Pc=34 bar and Tc=−147° C.) and mixtures containing these compounds/elements. Pc stands for critical pressure and Tc for critical temperature. Compounds according to group (b) are typically selected amongst conventional liquids of the same general types as discussed for solvents above but with the difference that the PKI present in the solution must be poorly soluble in the antisolvent. Particular liquids of group (b) comprise methanol, ethanol, acetone water and mixtures containing one or more of these fluids.

The antisolvents of group (a) above are typically used at pressures and temperatures providing i) supercritical conditions (supercritical fluid) or ii) a subcritical conditions (subcritical fluid) in the particle formation function and/or upstream of this function, such as in the mixing function and upstream of this latter function.

Variant (i) means pressures and temperatures which are typically above the critical pressure Pc and critical temperature Tc of the antisolvent used. For the pressure this typically means pressures in the interval (1.0-7.0)×Pc or in the interval ≧10 bar, suitably ≧20 bar with preference for ≧30 bar, higher than Pc with illustrative upper limits being 100 bar, 200 bar and 300 bar higher than Pc. For the temperature this typically means temperatures within (1.0-4.0)×Tc or in the interval of ≧5° C., suitably ≧10° C. with preference for ≧15° C. above Tc with illustrative upper limits being 10° C., 40° C. and 50° C. above Tc.

Variant (ii) means that at least one of temperature and pressure, with preference for only the temperature, is/are below the critical value. (Tc and Pc, respectively). Thus the temperature may be in the interval of (0.1-1)×Tc, such as (0.5-1)×Tc, or lower. Further, the temperature may be low, such as −10° C. or −30° C. These temperatures may be combined with pressures as defined in the preceding paragraph or with pressures lower than the Pc of the used antisolvent. For carbon dioxide this means that the temperature in the particle formation function is <+31° C., such as about +25° C. or lower combined with a pressure above or below 74 bar.

The antisolvents of group (b) above are typically used in the subcritical state, i.e. as a subcritical fluid.

For methods of the invention utilizing subcritical conditions in the particle formation chamber. This include that the pressure in the mixing function and in the antisolvent fluid always is higher than in the particle formation function.

In one aspect of the invention, there is provided a method of producing stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, comprising

-   -   a) providing a first, preferably pressurized stream of said         protein kinase inhibitor dissolved in a solvent;     -   b) providing a second, preferably pressurized stream of         antisolvent; wherein said at least one polymeric stabilizing and         matrix-forming component is present in either said first or         second stream; and     -   c) mixing said first and second streams, and spraying the mixed         stream at the outlet of a nozzle, whereby said hybrid         nanoparticles are formed; followed by collecting said hybrid         nanoparticles.

In one embodiment of this aspect, said polymeric stabilizing and matrix-forming component is present in said solvent.

In another embodiment of this aspect, said polymeric stabilizing and matrix-forming component is present in said antisolvent.

In another embodiment of this aspect, at least one of said fluid streams is a supercritical fluid stream, preferably a super- or subcritical CO₂ fluid stream.

In another embodiment of this aspect, said stream of antisolvent is a super- or subcritical CO₂ fluid stream.

In another embodiment of this aspect, said stream of solvent is a super- or subcritical CO₂ fluid stream.

In another embodiment of this aspect, said at least one polymeric stabilizing and matrix-forming component is present in said super- or subcritical CO₂ fluid stream.

In another embodiment of this aspect, said stream of solvent is a subcritical CO₂ fluid stream. Said super- or subcritical CO₂ fluid stream is preferably provided at about 25° C. or lower, at a pressure of from about 100 to about 150 bar. Said stream of solvent may, however, comprise e.g. nitrogen, methane, ethane, propane, ethylene, methanol, ethanol, acetone, water or a mixture of these compounds such as a mixture of CO₂ and nitrogen. Moreover a further solvent, such as an organic solvent (e.g. methanol, acetone) or aqueous solution (e.g. water) may be added as modifier into a super-/sub-critical solvent.

In another embodiment of this aspect, said stream of antisolvent is a super- or subcritical CO₂ fluid stream. Said super- or subcritical CO₂ fluid stream is preferably provided at about 25° C. or lower, at a pressure of from about 100 to about 150 bar. Said stream of antisolvent may, however, comprise e.g. nitrogen, methane, ethane, propane, ethylene, methanol, ethanol, acetone, water or a mixture of these compounds such as a mixture of CO₂ and nitrogen. Moreover a solvent, such as an organic solvent (e.g. methanol, acetone) or aqueous solution (e.g. water) may be added as modifier into a super- or subcritical antisolvent.

The polymeric stabilizing and matrix-forming component of the present in the methods of the invention includes, but not limited to, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (e.g. HPC ef, HPC If and HPC jf), hydroxypropyl methylcellulose (e.g. Methocel E3 and E15 and Pharmacoat), hydroxypropyl methylcellulose acetate succinate (HPMC AS), hydroxypropyl methylcellulose phthalate (e.g. HPMCP HP55), polyvinylpyrrolidone (e.g. PVP 30K and PVP 90K), polyvinyl acetate phthalate (PVAP), copolyvidone (e.g. Kollidon VA 64), crospovidon (e.g. Kollidon CL), methacrylic acid and ethylacrylate copolymer (e.g. Kollicoat ME), methacrylate acid and methyl methacrylate copolymer (e.g. Eudragit L100), polyethylene glycol (PEG), DL lactide/glycolide copolymer, poly DL-lactide, cellulose acetate phthalate (CAP), carbomer homopolymer Type A (Carbopol 971P), carbomer homopolymer Type B (Carbopol 974P), aminoalkyl methacrylate copolymers (e.g. Eudragit RL100, RL PO or RS PO) and Poloxamers (e.g. Pluronics, Kolliphor).

Consequently, in another embodiment of this aspect, said polymeric stabilizing and matrix-forming component is selected from methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, polyvinylpyrrolidone, polyvinyl acetate phthalate, copolyvidone, crospovidon, methacrylic acid and ethylacrylate copolymer, methacrylate acid and methyl methacrylate copolymer, polyethylene glycol, DL lactide/glycolide copolymer, poly DL-lactide, cellulose acetate phthalate, carbomer homopolymer Type A, carbomer homopolymer Type B, aminoalkyl methacrylate copolymers, and Poloxamers. Preferably, said polymeric stabilizing and matrix-forming component is selected from hydroxypropyl methylcellulose phthalate, hydroxypropyl cellulose, copolyvidon, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate and polyvinylpyrrolidone.

In another embodiment of this aspect, a solubilizer is added to the hybrid nanoparticles obtained in step c. In this context, the solubilizer will be present separately from the particles. Said solubilizer may be selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate and a hydrogenated castor oil, such as PEG-40 hydrogenated castor oil or PEG-35 hydrogenated castor oil. Furthermore, said solubilizer may be a poloxamer.

The methods of the invention provide stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, which display increased dissolution rate.

Consequently, in another embodiment of this aspect, there is provided method of producing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, wherein said hybrid nanoparticles display an increased dissolution rate of said protein kinase inhibitor, compared to the dissolution rate of said protein kinase inhibitor in raw, crystalline form.

Typically, said dissolution rate is measured by a flow through cell system in sink conditions, e.g., according to the US Pharmacopea (USP4). Dissolution measurement in sink conditions of hybrid nanoparticles may be measured in a method consisting of adding the wished amount of powder into a flow through cell system (SOTAX, Allschwill, Switzerland), mounting the cell onto its apparatus and then pumping the appropriate medium (typically FaSSIF, FeSSIF, SGF) through the powder. The temperature of the apparatus is typically set to 37° C. The amount of powder added into the cell depends on drug load of the powder: The exact amount of powder can be calculated from results obtained from drug load analysis of the powders. The PKI may be added into the flow through cell and a flow rate between 5 and 25 ml medium/min is pumped through the powder. One ml samples of the medium passing through the cell is collected at predetermined times and subsequently analyzed by HPLC (e.g. C18 column Eclipse, 4.6 mm×15 cm, 1 ml/min, detection 254 to 400 nm). Samples are typically taken after 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 and 40 min from the moment the medium comes out from the flow through cell. The accumulated % solubilized of the amount of active substance added into the flow through cell can be calculated and plotted against time (min). The initial slope (“initial dissolution rate”, representing 0-10 minutes) of the graph may be estimated and taken as the dissolution rate of the material in sink condition at 37° C. in the given dissolution medium.

Preferably, the dissolution rate is measured within the initial 0 to 10 minutes of dissolution.

The increased dissolution rate is preferably measured in a solution as a dissolution rate ratio of said hybrid nanoparticles and said protein kinase inhibitor in raw, crystalline form. Preferably said ratio is from about 1.5:1 to about 500:1, such as from about 10:1 to about 30:1.

Preferably, the dissolution rate is measured in a solution with intestinal pH, such as FaSSIF or FeSSIF or in a solution with gastric pH, such as SGF.

Typically, said dissolution rate is measured by a flow through cell system, for instance in sink conditions. Dissolution measurement in sink conditions of hybrid nanoparticles may be measured in a method consisting of adding the wished amount of powder into a flow through cell system (SOTAX, Allschwill, Switzerland), mounting the cell onto its apparatus and then pumping the appropriate medium (typically FaSSIF, FeSSIF, SGF) through the powder. The temperature of the apparatus is typically set to 37° C. The amount of powder added into the cell depends on drug load of the powder: The exact amount of powder can be calculated from results obtained from drug load analysis of the powders. The PKI may be added into the flow through cell and a flow rate between 5 and 25 ml medium/min is pumped through the powder. One ml samples of the medium passing through the cell is collected at predetermined times and subsequently analyzed by HPLC (e.g. C18 column Eclipse, 4.6 mm×15 cm, 1 ml/min, detection 254 to 400 nm). Samples are typically taken after 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 and 40 min from the moment the medium comes out from the flow through cell. The accumulated % solubilized of the amount of active substance added into the flow through cell, can be calculated and plotted against time (min). The initial slope (“initial dissolution rate”, representing 0-10 minutes) of the graph can be estimated and taken as the dissolution rate of the material in sink condition at 37° C. in the given dissolution medium.

In another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, which produces particles that provides a solubility increase of inhibitor in a solution, said increase measured as the area under the curve (AUC) during about from 40 minutes to about 90 minutes, in said solution as compared with the AUC of inhibitor in raw, crystalline form. Preferably, said increase is from about 2:1 to about 10 000:1, wherein 1 represents AUC of inhibitor in raw, crystalline form. Preferably, said increase is measured in a solution with gastric pH, such as SGF, or measured in a solution with intestinal pH, such as FaSSIF or FeSSIF. The methods may produce particles that provide a solubility increase of inhibitor in a solution up to supersaturation, said increase measured as the area under the curve (AUC) during about from 40 minutes to about 90 minutes, in said solution as compared with the AUC of inhibitor in raw, crystalline form.

In another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, characterized by providing an amorphous powder X-ray diffraction pattern.

In another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, wherein the dissolution rate of said stable, amorphous hybrid nanoparticles remain stable to at least about 90%, after 6 months of storage or more, at room temperature.

In another embodiment of this aspect, said protein kinase inhibitor is a tyrosine kinase inhibitor selected from the group consisting of lapatinib, pazopanib, nilotinib, erlotinib, dasatinib, gefitinib, sorafenib, crizotinib, vemurafenib and axitinib; or salts or hydrates or solvates thereof, or combinations thereof. In some embodiments it may be advantageous to use other PKIs. Examples of PKI include, but are not limited to afatinib, bosutinib, cediranib, fostamatinib, imatinib, lenvatinib, lestaurtinib, motesanib, mubritinib, pegaptanib, ruxolitinib, semaxanib, sunitinib, tandunitib, tipifamib and vandetanib; or salts or hydrates or solvates thereof, or combinations thereof.

In another embodiment of this aspect, said hybrid nanoparticles has an average particle diameter size of less than about 1000 nm, such as less than about 500 nm, preferably less than 250 nm.

In another embodiment of this aspect, said solvent is an organic solvent selected from DMSO and trifluoroethanol, or a mixture of these solvents, or mixture of these solvents with other organic solvent such as DMSO/acetone, DMSO/tetrahydrofurane or trifluoroethanol/ethyl acetate.

In another embodiment of this aspect, said particles further comprise a solubilizer within the particles. Said solubilizer may be polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate, or a hydrogenated castor oil, such as PEG-40 hydrogenated castor oil or PEG-35 hydrogenated castor oil.

In another embodiment of this aspect, the method further comprises formulating the particles retained in step (c) as a pharmaceutical composition containing the particles and optionally further pharmaceutically acceptable excipients.

In another aspect of the invention, there is provided stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, obtainable by the methods described above.

The hybrid nanoparticles produced by the methods of the present invention may also dissolve and the protein kinase inhibitor may be systemically absorbed independently of the pH in the surrounding environment, and typically approximately in equal amounts, especially at both a gastric pH, such as from about pH 1.2 to about pH 2.1, preferably about 1.7 and at a intestinal pH such as from about pH 4.5 to about pH 8, preferably at a pH of about 6. With systemically absorbed, is meant that the protein kinase inhibitor is released from the hybrid nanoparticles and taken up by the systemic blood stream. Therefore, in another embodiment of this aspect, there is provided a method, wherein said protein kinase inhibitor is systemically absorbed independently of the pH. Typically, said protein kinase inhibitor is systemically absorbed with approximately equal amounts at both a gastric pH and at an intestinal pH. Preferably, said acid pH is about pH 1.4 and preferably said neutral pH is about pH 6.5.

With approximately equal amounts is meant that the concentration of protein kinase inhibitor in the blood stream, after exposure is approximately similar. This may be illustrated by a ratio, wherein the concentration of protein kinase inhibitor in the blood stream is measured after administration in gastric pH conditions (A) and compared with the concentration of protein kinase inhibitor in the blood stream is measured after administration in intestinal pH conditions (N). Typically, the ratio A:N is from about 0.75:1 to about 1.5:1 and preferably from about 1:1 to about 1.25:1. The concentration measurement of protein kinase inhibitor in the blood stream may be carried out as an area under the curve (AUC) during 0-24 hours, the maximum concentration (Cmax) or as bioavailability.

Consequently, in another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, wherein the concentration of systemically absorbed protein kinase inhibitor in gastric pH conditions compared with the concentration of systemically absorbed protein kinase inhibitor in intestinal pH conditions is in a ratio of from about 0.75:1 to about 1.5:1, preferably of from about 1:1 to about 1.25:1. Typically said gastric pH condition represents a pH of about 1.4 and said intestinal pH condition represents a pH of about 6. Typically, the concentration is measured as area under the curve (AUC) during 0-24 hours of exposure of the composition or as the maximum concentration (Cmax).

The amounts of systemically absorbed protein kinase inhibitor may be measured in various ways. There is provided, in Example 14 in the present disclosure, a method for measurement of systemically absorbed protein kinase inhibitors at various pHs, i.e. under both acid and neutral conditions.

In another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, which produces a solubility increase of inhibitor in a solution up to supersaturation, said increase measured as the area under the curve (AUC) during about 90 minutes, in said solution and compared with the AUC of inhibitor in crystalline form. Said increase may be from to about 2:1 to about 1000:1, wherein 1 represents AUC of inhibitor in crystalline form.

For understanding how the hybrid nanoparticles produced by the methods of the invention will dissolve in vivo in the different environments of the stomach, small intestine, large intestine and colon, it is important to choose an appropriate solution for in vitro dissolution testing. It is critical that the in vitro test conditions mimic the in vivo environment as closely as possible, for example pH and osmolarity. Typically, for intestinal uptake, the pH is between 6 and 7. Therefore, the solution may hold a pH from about pH 6 to about pH 7, such as about pH 6.5.

Therefore, in embodiments of the invention, the solution for testing has a pH from about pH 4.5 to about pH 8, such as about pH 6.5 or such as about pH 5. The solutions may represent Fasted Simulated State Intestinal Fluid (FaSSIF) or Fed Simulated State Intestinal Fluid (FeSSIF).

Typically, for gastric uptake, the pH is between 1 and 2. Therefore, the solution may hold a pH from about pH 1 to about pH 2, such as about pH 1.4. Therefore, in embodiments of the invention, the solution for testing may represent Simulated Gastric Fluid (SGF).

The choice of solution will be dependent on where in the intestinal tract and under what conditions (fasted or fed) the composition is desired to dissolve and be taken up. Recipes and preparation of these solutions are obtainable from the manufacturer (Biorelevant, Croydon, U.K.). Further details are also disclosed in Jantratid, E., and Dressman, J. (2009) Dissolut. Technol. 8, 21-25).

In another embodiment of this aspect, there is provided a method of producing stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component and further a solubilizer selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate, Poloxamers (e.g. Pluronics, Kolliphor) and a hydrogenated castor oil, such as PEG-40 hydrogenated castor oil or PEG-35 hydrogenated castor oil. Preferably said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer or a hydrogenated castor oil, such as PEG-40 hydrogenated castor oil or PEG-35 hydrogenated castor oil.

It may further be advantageous to use different dispersion agents and/or other excipients to achieve different dissolution profiles. For example using Nilotinib HCl powder with HPC EF, HPMC (e.g. Methocel) and poloxamer (e.g. Lutrol® F127) result in hybrid nanoparticles with different dissolution profiles.

The amount of PKI in the hybrid nanoparticles produced by the methods of the present invention may be less or more, such as wherein the amount of PKI in the hybrid nanoparticles is from about 0.01% by weight to about 99.9% by weight.

In another embodiment of this aspect, there is provided hybrid nanoparticles produced by the methods of the present invention, wherein the amount of PKI in the hybrid nanoparticles is from about 10% by weight to about 70% by weight.

In another embodiment of this aspect, there is provided hybrid nanoparticles produced by the methods of the present invention, wherein the amount of PKI in the hybrid nanoparticles is from about 10% by weight to about 50% by weight.

In some embodiments, it may be advantageous that the amount of PKI in the hybrid nanoparticles is from 5% by weight to about 50% by weight, from 10% by weight to about 40% by weight, from about 10% by weight to about 30% by weight, or from about 10% by weight to about 20% by weight.

Control of the characteristics of the particles may be convenient for specific applications. Particle size, particle agglomeration, particles porosity and the choice and ratio of the polymeric stabilizing and matrix-forming agent could be modified in order to increase or decrease the surface area to volume ratio of the particle or behaviour of the particles in a gastroinstestinal fluids, leading to an increase or decrease of the dissolution rate. Dependent on the desired dissolution characteristics such particles characteristics may be adapted. Furthermore, particles with different characteristics may be present in the same pharmaceutical composition to provide an initial dose and a prolonged or delayed dose of active ingredient. Additionally, it may be advantageous to provide different PKIs and/or other active ingredient(s) in different primary particles with different characteristics adapted to provide desired dissolution rates for each active ingredient(s).

Other embodiments of the invention provide pharmaceutical compositions comprising the hybrid nanoparticles obtainable by the methods of the invention. Such compositions may further comprise at least one pharmaceutically acceptable solubilizer. Said solubilizer may be present separated from the hybrid nanoparticles in the composition or be randomly intermixed with the hybrid nanoparticles in the pharmaceutical composition. The pharmaceutical composition may also be in a dosage form consisting of several layers, for example laminated or multilayered tablets, such that the hybrid nanoparticles are separated from the solubilizer. The solubilizer may be selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate and a hydrogenated castor oil, such as PEG-40 hydrogenated castor oil or PEG-35 hydrogenated castor oil. Said solubilizer may also be a poloxamer.

In another embodiment of this aspect, there is provided stable, amorphous hybrid nanoparticles, comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, obtainable by methods of the present invention.

In another embodiment of this aspect, there is provided a method of the present invention, further comprising formulating the particles retained in step (c) as a pharmaceutical composition containing the particles and optionally further pharmaceutically acceptable excipients.

It will be appreciated that the amount of a protein kinase inhibitor in the hybrid nanoparticles produced by the methods of the present invention required for use in treatment will vary not only with the particular inhibitor selected but also with the route of administration, the nature of the condition for which treatment is required and the age, weight and condition of the patient and will be ultimately at the discretion of the attendant physician. In general however a suitable dose may be in the range of from about 0.005 to about 30 mg/kg of body weight per day, preferably in the range of 0.05 to 10 mg/kg/day.

The desired dose is conveniently presented in a single dose or as a divided dose administered at appropriate intervals, for example as two, three, four or more doses per day. Dependent on the need of the treatment and/or prevention, the desired dose may also be, for example, once every two days, once every three days, or even once a week.

The composition is conveniently administered in unit dosage form; for example containing 0.5 to 1500 mg, conveniently 1 to 1000 mg, most conveniently 5 to 700 mg of active ingredient per unit dosage form. The compositions of the invention will normally be administrated via the oral, parenteral, intravenous, intramuscular, subcutaneous or other injectable ways, buccal, rectal, vaginal, transdermal and/or nasal route and/or via inhalation, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.

Pharmaceutical compositions include but are not limited to those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compositions may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. Pharmaceutical compositions suitable for oral administration are conveniently presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active substance.

Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art.

The compositions may be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing and/or dispersing agents.

The above described compositions may be adapted to give sustained release of the active inhibitor.

The following examples are provided to illustrate various embodiments of the present invention and shall not be considered as limiting in scope.

EXAMPLES

Below follows a number of non-limiting examples of hybrid nanoparticles produced by the methods of the present invention. In the tables, the following abbreviations to “compositions” apply:

“I” represents the protein kinase inhibitor (PKI);

“P” represents the polymeric stabilizing and matrix-forming component;

“S” represents the solubilizer;

“I+P” represents a physical mix of the inhibitor with the polymeric stabilizing and matrix-forming component, i.e. without further processing;

“I+S” represents a physical mix of the inhibitor with the solubilizer;

“I+P+S” represents a physical mix of the inhibitor, the polymeric stabilizing and matrix-forming component and the solubilizer;

“I/P” represents hybrid nanoparticles with the inhibitor and the polymeric stabilizing and matrix-forming component;

“I/P+S” represents hybrid nanoparticles with the inhibitor and the polymeric stabilizing and matrix-forming component and a separate solubilizer added;

“I/P/S” represents hybrid nanoparticles with the inhibitor, the polymeric stabilizing and matrix-forming component and the solubilizer.

“Exp” represents the experiment number.

The hybrid nanoparticles were produced with exemplary PKIs, polymeric stabilizing and matrix-forming components (“Polymers”), solubilizers, solution concentrations, ratios, solvents, antisolvents, temperatures and pressures as set out below and in Table A.

A 3-6% w/v PKI/polymer solution in solvent, with a ratio PKI/polymer of about 20-70% w/w, was pumped through XSpray's RightSize nozzle at the flow rate of 1 ml/min using a high-performance liquid chromatography pump, together with a 100 g/min CO₂ (super- or subcritical) stream. The pressure in the precipitation chamber was set to about 100-175 bar and the temperature was set to about 10 to 50° C. Both streams contact within the nozzle and the hybrid nanoparticles were formed and subsequently collected in the particle in the collecting chamber. The scCO₂ and solvent passed through the filtering system of the collecting chamber and were drained via the back pressure regulator outlet which maintains the pressure within the precipitation and collecting chambers. After pumping of the PKI/polymer solution and cleaning of the tubing with the same solvent used to prepare the PKI/polymer solution, residual solvents left within both the precipitation and collecting chambers were removed by flushing these chambers with pure CO₂. After the flushing process, the CO₂ was slowly drained off from the collecting chamber. Once the CO₂ had been completely removed, the particles on the filtering system were collected for analysis.

For I/P/S type particles, a defined amount of solubilizer is added and dissolved into the PKI/polymer solution before pumping the solution through the nozzle for precipitation according to the methods described above.

For I/P+S type particles, a defined amount of solubilizer is added to the hybrid nanoparticles in a glass vial. The glass vial is slowly rotated for mixing of the solubilizer with the hybrid nanoparticles.

TABLE A Stable, amorphous hybrid nanoparticles with exemplary PKIs, polymeric stabilizing and matrix-forming components, solvents, antisolvents and conditions. Solution Ratio conc. % PKI/Polymer Solvent Temperature PKI/Polymer Exp. # (w/v) % (w/w) & Antisolvent & Pressure Axitinib/ 160, 162 & 5% 25% DMSO 25° C. Kollidon 581 & CO₂ & 125 Bars VA64 Crizotinib/ 153, 155, 5% 25% DMSO 25° C. PVP 30K 156 & 571 & CO₂ & 125 Bars Dasatinib/ 140, 141 & 4% 35% DMSO/Acetone 15° C. Kollidon 551 (1:2) & CO₂ & 125 Bars VA64 Erlotinib HCl/ 511 3.6%   35% TFE 25° C. HPMC AS & CO₂ & 150 Bars Gefitinib/ 135, 137 & 4% 35% DMSO/Acetone 40° C. HPMCP HP55 541 (1:2) & CO2 & 150 Bars Lapatinib base/ 531 5% 66% DMSO/Acetone 40° C. HPC If (1:2) & CO2 & 150 Bars Nilotinib base/ 501 5% 40% TFE 15° C. HPMCP HP55 & CO₂ & 125 Bars Pazopanib HCl/ 521 3.6%   35% TFE 25° C. PVP 90K & CO₂ & 150 Bars Sorafenib 561 4% 35% DMSO/Acetone 40° C. tosylate/ (1:2) & CO2 & 150 Bars HPMCP HP55 Vemurafenib/ 168, 170 & 5% 25% DMSO 25° C. CAP 592 & CO₂ & 125 Bars

General Description of Dissolution Measurement Assay

The method consists of adding the wished amount of powder of hybrid nanoparticles into a glass vial and then pouring in it the appropriate medium (typically FaSSIF, FeSSIF or SGF). The medium was prepared in accordance with the manufacturer's instructions. The amount of powder added depends on the wished “total PKI concentration”. For some experiments where powders with high drug loads were tested and compared, the real amount of PKI in the hybrid nanoparticles was not taken in account. For other experiments, the drug load was first estimated by HPLC and the amount of powder to obtain the drug concentration was calculated.

Typically, the powder was added in a 8 mL glass bottle and 7 mL of solution was added (typically FaSSIF, FeSSIF or SGF). The glass bottle was put on a shaker (approximately 1 rotation per minute) for dissolution. Samples of 500 μl where taken after different times, and subsequently centrifuged at approximately 15000 g for 3 minutes. The resulting supernatant was then analyzed by HPLC (C₁₈ column Eclipse, 4.6 mm×15 cm, 1 mL/min, detection at 254-400 nM. Generally samples were taken after 5, 30 and 90 min and eventually 150 min.

Example 1 Stable, Amorphous Hybrid Nanoparticles with Nilotinib—Solubility at pH 6.5 and pH 5

A number of experiments were carried out, wherein nilotinib base or nilotinib HCl represented the protein kinase inhibitor. The experiments were carried out by measuring concentration of solubilized PKI (mg/L) after 5, 30 and 90 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Further, experiments were carried out in an alternative solution at about pH 5, namely FeSSIF (Fed State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of protein kinase inhibitor was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 1 and 2, where Table 1 provides data of concentration of nilotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution, whereas Table 2 provides data of % solubilized nilotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 90 minutes dissolution and the AUC increase of hybrid nanoparticles, compared to nilotinib HCl in raw, crystalline form added to the solution (experiments 1-40). In Tables 3 and 4, there is provided dissolution data in FeSSIF solution, presented similarly as Table 1 and 2 (experiments 41-55). Table 5 provides data from a comparative experiment with similar hybrid nanoparticles, carried out in FaSSIF and FeSSIF, respectively (experiments 56-57). Table 6 presents further comparative data for experiments carried out in FaSSIF and FeSSIF, respectively, with hybrid nanoparticles produced by the methods of the invention.

TABLE 1 Nilotinib - concentration of nilotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 1 I Nilotinib HCl 100 — — 0.1 0.2 0.1 (raw) 100 mg 2 I Nilotinib HCl 100 — — 0.2 0.2 0.2 (raw) 500 mg 3 I Nilotinib HCl 100 — — 0.2 0.3 0.2 (raw) 1000 mg 4 I Nilotinib Base 100 — — 0.6 0.5 0.2 (raw) 500 mg 5 I + P Nilotinib HCl 100 HPMCP HP55 — 0.2 0.5 0.5 (raw) 1000 mg 2000 mg 6 I + P Nilotinib HCl 100 PVAP — 1.3 0.2 0.4 (raw) 1000 mg 2000 mg 7 I + P Nilotinib HCl 100 Eudragit L100 — 0.2 0.4 0.2 (raw) 1000 mg 2000 mg 8 I + P Nilotinib HCl 100 Methocel E15 — 0.1 0.1 0.1 (raw) 1000 mg 2000 mg 9 I + S Nilotinib HCl 100 — Soluplus 0.4 0.3 0.4 (raw) 1000 mg 357.5 mg 10 I + S Nilotinib HCl 100 — Soluplus 0.4 0.5 0.5 (raw) 1000 mg 715 mg 11 I + S Nilotinib HCl 100 — Soluplus 0.4 0.5 0.6 (raw) 1000 mg 1072 mg 12 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.4 0.6 1.0 (raw) 500 mg 750 mg 715 mg 13 I + P + S Nilotinib HCl 100 PVAP Soluplus 0.2 0.2 0.3 (raw) 500 mg 750 mg 715 mg 14 I + P + S Nilotinib HCl 100 HPMCP HP55 TPGS 0.5 0.9 1.1 (raw) 500 mg 750 mg 1000 mg 15 I + P + S Nilotinib HCl 100 PVAP TPGS 0.2 0.4 0.5 (raw) 500 mg 750 mg 1000 mg 16 I + P + S Nilotinib Base 100 HPMCP HP55 Soluplus 0.2 0.5 0.4 (raw) 500 mg 750 mg 715 mg 17 I/P Nilotinib HCl 50 HPMCP HP55 — 9.5 5.6 4.5 100 mg 100 mg 18 I/P Nilotinib HCl 40 HPMCP HP55 — 10.4 5.0 3.7 100 mg 150 mg 19 I/P Nilotinib HCl 50 PVAP — 7.3 5.0 4.1 100 mg 100 mg 20 I/P Nilotinib HCl 40 PVAP — 8.7 5.0 3.4 100 mg 150 mg 21 I/P Nilotinib HCl 50 Methocel E15 — 1.4 1.5 1.8 100 mg 100 mg 22 I/P Nilotinib HCl 50 Eudragit L100 — 5.1 5.9 4.9 100 mg 100 mg 23 I/P Nilotinib Base 40 HPMCP HP55 — 9.7 4.7 3.8 100 mg 150 mg 24 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 53.4 46.1 35.6 500 mg 500 mg 715 mg 25 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 85.9 87.9 80.8 500 mg 500 mg 1430 mg 26 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 117.0 127.1 116.9 500 mg 500 mg 2145 mg 27 I/P + S Nilotinib HCl 50 HPMCP HP55 TPGS 49.6 30.1 22.3 500 mg 500 mg 1000 mg 28 I/P + S Nilotinib HCl 50 HPMCP HP55 TPGS 98.4 57.4 42.6 500 mg 500 mg 2000 mg 29 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 93.5 45.2 14.1 500 mg 750 mg 357.5 mg 30 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 145.0 134.3 36.8 500 mg 750 mg 715 mg 31 I/P + S Nilotinib HCl 40 HPMCP HP55 TPGS 93.8 31.0 22.4 500 mg 750 mg 1000 mg 32 I/P + S Nilotinib HCl 40 PVAP Soluplus 82.9 137.9 42.9 500 mg 750 mg 715 mg 33 I/P + S Nilotinib HCl 40 PVAP TPGS 77.8 32.3 22.8 500 mg 750 mg 1000 mg 34 I/P + S Nilotinib HCl 50 Methocel E15 Soluplus 3.3 4.0 5.8 500 mg 500 mg 715 mg 35 I/P + S Nilotinib HCl 50 Methocel E15 TPGS 4.8 5.4 6.7 500 mg 500 mg 1000 mg 36 I/P + S Nilotinib Base 40 HPMCP HP55 Soluplus 178.1 120.4 33.7 500 mg 750 mg 715 mg 37 I/P/S Nilotinib HCl 25.4 HPMCP HP55 Soluplus 25.9 15.8 16.3 500 mg 750 mg 715 mg 38 I/P/S Nilotinib HCl 25.4 PVAP Soluplus 9.5 13.2 10.1 500 mg 750 mg 715 mg 39 I/P/S Nilotinib HCl 22.2 HPMCP HP55 TPGS 16.2 13.7 3.9 500 mg 750 mg 1000 mg 40 I/P/S Nilotinib HCl 22.2 PVAP TPGS 13.3 12.1 9.7 500 mg 750 mg 1000 mg

TABLE 2 Percentage solubilized nilotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase of stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to nilotinib HCl in raw, crystalline form added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 1 I Nilotinib HCl 100 — — 0.20 13.0 — (raw) 100 mg 2 I Nilotinib HCl 100 — — 0.04 23.5 — (raw) 500 mg 3 I Nilotinib HCl 100 — — 0.03 21.8 — (raw) 1000 mg 4 I Nilotinib Base 100 — — 0.50 36.5 — (raw) 500 mg 5 I + P Nilotinib HCl 100 HPMCP HP55 — 0.05 39.3 2.0 (raw) 1000 mg 2000 mg 6 I + P Nilotinib HCl 100 PVAP 2000 mg — 0.02 40.0 2.1 (raw) 1000 mg 7 I + P Nilotinib HCl 100 Eudragit L100 — 0.04 26.0 1.3 (raw) 1000 mg 2000 mg 8 I + P Nilotinib HCl 100 Methocel E15 — 0.01 8.8 0.5 (raw) 1000 mg 2000 mg 9 I + S Nilotinib HCl 100 — Soluplus 0.03 30.8 1.6 (raw) 1000 mg 357.5 mg 10 I + S Nilotinib HCl 100 — Soluplus 0.05 42.3 2.2 (raw) 1000 mg 715 mg 11 I + S Nilotinib HCl 100 — Soluplus 0.05 45.3 2.3 (raw) 1000 mg 1072 mg 12 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.12 61.5 3.2 (raw) 500 mg 750 mg 715 mg 13 I + P + S Nilotinib HCl 100 PVAP Soluplus 0.04 20.5 1.1 (raw) 500 mg 750 mg 715 mg 14 I + P + S Nilotinib HCl 100 HPMCP HP55 TPGS 0.18 78.8 4.1 (raw) 500 mg 750 mg 1000 mg 15 I + P + S Nilotinib HCl 100 PVAP TPGS 0.08 35.0 1.8 (raw) 500 mg 750 mg 1000 mg 16 I + P + S Nilotinib Base 100 HPMCP HP55 Soluplus 0.10 36.3 1.9 (raw) 500 mg 750 mg 715 mg 17 I/P Nilotinib HCl 50 HPMCP HP55 — 5.6 515.5 26.6 100 mg 100 mg 18 I/P Nilotinib HCl 40 HPMCP HP55 — 5.0 479.5 24.7 100 mg 150 mg 19 I/P Nilotinib HCl 50 PVAP — 5.0 445.0 22.9 100 mg 100 mg 20 I/P Nilotinib HCl 40 PVAP — 5.0 445.0 22.9 100 mg 150 mg 21 I/P Nilotinib HCl 50 Methocel E15 — 1.5 138.8 7.2 100 mg 100 mg 22 I/P Nilotinib HCl 50 Eudragit L100 — 5.9 474.3 24.2 100 mg 100 mg 23 I/P Nilotinib Base 40 HPMCP HP55 — 4.7 459.3 23.7 100 mg 150 mg 24 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 9.2 3828.3 197.3 500 mg 500 mg 715 mg 25 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 17.6 7448.3 383.9 500 mg 500 mg 1430 mg 26 I/P + S Nilotinib HCl 50 HPMCP HP55 Soluplus 25.4 10663.8 549.7 500 mg 500 mg 2145 mg 27 I/P + S Nilotinib HCl 50 HPMCP HP55 TPGS 6.0 2692.3 138.8 500 mg 500 mg 1000 mg 28 I/P + S Nilotinib HCl 50 HPMCP HP55 TPGS 11.5 5193.5 267.7 500 mg 500 mg 2000 mg 29 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 9.0 3746.5 193.1 500 mg 750 mg 357.5 mg 30 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 26.9 8974.8 462.6 500 mg 750 mg 715 mg 31 I/P + S Nilotinib HCl 40 HPMCP HP55 TPGS 6.2 3396.5 175.1 500 mg 750 mg 1000 mg 32 I/P + S Nilotinib HCl 40 PVAP Soluplus 27.6 8391.3 432.5 500 mg 750 mg 715 mg 33 I/P + S Nilotinib HCl 40 PVAP TPGS 6.5 3223.8 166.2 500 mg 750 mg 1000 mg 34 I/P + S Nilotinib HCl 50 Methocel E15 Soluplus 0.8 393.5 20.3 500 mg 500 mg 715 mg 35 I/P + S Nilotinib HCl 50 Methocel E15 TPGS 1.1 505.5 25.9 500 mg 500 mg 1000 mg 36 I/P + S Nilotinib Base 40 HPMCP HP55 Soluplus 24.1 8799.5 453.6 500 mg 750 mg 715 mg 37 I/P/S Nilotinib HCl 25.4 HPMCP HP55 Soluplus 3.2 1549.0 79.8 500 mg 750 mg 715 mg 38 I/P/S Nilotinib HCl 25.4 PVAP Soluplus 2.6 1006.5 51.9 500 mg 750 mg 715 mg 39 I/P/S Nilotinib HCl 22.2 HPMCP HP55 TPGS 2.7 942.3 48.6 500 mg 750 mg 1000 mg 40 I/P/S Nilotinib HCl 22.2 PVAP TPGS 2.4 1004.8 51.8 500 mg 750 mg 1000 mg

TABLE 3 Nilotinib - concentration of nilotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 41 I Nilotinib HCl 100 — — 0.6 0.9 0.9 (raw) 500 mg 42 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.4 0.6 1.0 (raw) 500 mg 750 mg 715 mg 43 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.2 0.2 0.3 (raw) 500 mg 750 mg 1000 mg 44 I + P + S Nilotinib HCl 100 PVAP Soluplus 0.5 0.9 1.1 (raw) 500 mg 750 mg 715 mg 45 I + P + S Nilotinib HCl 100 PVAP TPGS 0.2 0.4 0.5 (raw) 500 mg 750 mg 1000 mg 46 I/P Nilotinib HCl 40 HPMCP HP55 — 16.2 45.6 63.3 500 mg 750 mg 47 I/P Nilotinib HCl 40 PVAP — 3 7.7 11.2 500 mg 150 mg 48 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 47.7 85.5 109.4 500 mg 750 mg 715 mg 49 I/P + S Nilotinib HCl 40 HPMCP HP55 TPGS 74.8 112.4 125.5 500 mg 750 mg 1000 mg 50 I/P + S Nilotinib HCl 40 PVAP Soluplus 12.9 21.3 27.3 500 mg 750 mg 715 mg 51 I/P + S Nilotinib HCl 40 PVAP TPGS 20.5 29.8 31.8 500 mg 750 mg 1000 mg 52 I/P/S Nilotinib HCl 40 HPMCP HP55 Soluplus 42.3 81.5 108.1 500 mg 750 mg 715 mg 53 I/P/S Nilotinib HCl 40 HPMCP HP55 TPGS 86.3 116.3 128.8 500 mg 750 mg 1000 mg 54 I/P/S Nilotinib HCl 40 PVAP Soluplus 6.3 18.8 28.2 500 mg 750 mg 715 mg 55 I/P/S Nilotinib HCl 40 PVAP TPGS 20.5 29.8 31.8 500 mg 750 mg 1000 mg

TABLE 4 Percentage solubilized nilotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase of stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to nilotinib HCl in raw, crystalline form added to the FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 41 I Nilotinib HCl 100 — — 0.18 74.3 — (raw) 500 mg 42 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.12 61.5 0.8 (raw) 500 mg 750 mg 715 mg 43 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 0.04 20.5 0.3 (raw) 500 mg 750 mg 1000 mg 44 I + P + S Nilotinib HCl 100 PVAP Soluplus 0.18 78.8 1.1 (raw) 500 mg 750 mg 715 mg 45 I + P + S Nilotinib HCl 100 PVAP TPGS 0.08 35.0 0.5 (raw) 500 mg 750 mg 1000 mg 46 I/P Nilotinib HCl 40 HPMCP HP55 — 9.1 4080.0 54.9 500 mg 750 mg 47 I/P Nilotinib HCl 40 PVAP — 7.7 708.3 9.5 500 mg 150 mg 48 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 17.1 7631.3 102.8 500 mg 750 mg 715 mg 49 I/P + S Nilotinib HCl 40 HPMCP HP55 TPGS 22.5 9664.0 130.2 500 mg 750 mg 1000 mg 50 I/P + S Nilotinib HCl 40 PVAP Soluplus 4.3 1917.8 25.8 500 mg 750 mg 715 mg 51 I/P + S Nilotinib HCl 40 PVAP TPGS 6.0 2528.0 34.0 500 mg 750 mg 1000 mg 52 I/P/S Nilotinib HCl 40 HPMCP HP55 Soluplus 16.3 7341.3 98.9 500 mg 750 mg 715 mg 53 I/P/S Nilotinib HCl 40 HPMCP HP55 TPGS 23.3 10101.3 136.0 500 mg 750 mg 1000 mg 54 I/P/S Nilotinib HCl 40 PVAP Soluplus 3.8 1739.5 23.4 500 mg 750 mg 715 mg 55 I/P/S Nilotinib HCl 40 PVAP TPGS 6.0 2528.0 34.0 500 mg 750 mg 1000 mg

TABLE 5 Nilotinib - concentration of nilotinib HCl (mg/L) after 5, 30, 90 and 150 minutes dissolution in FaSSIF and FeSSIF solution, respectively. Drug Polymeric load stab. matrix. Conc Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 150 min 56 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 51.2 66 62.3 53.2 FaSSIFF 75 mg 112.5 mg 715 mg 57 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 24.8 43.1 50.7 53 FeSSIFF 75 mg 112.5 mg 715 mg

TABLE 6 Nilotinib - concentration of nilotinib HCl (mg/L) after 5, 30, 90 and 150 minutes dissolution in FaSSIF and FeSSIF solution, respectively presented as comparative data. Drug Polymeric load stab. matrix. Compare Compare Compare Compare ratio Component Solubilizer (%) (%) (%) (%) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 150 min  2 & 41 I Nilotinib HCl 100 — — 300 450 225 — (raw) 1000 mg 12 & 42 I + P + S Nilotinib HCl 100 HPMCP HP55 Soluplus 100 200 250 — (raw) 750 m g 715 mg 500 mg 18 & 46 I/P Nilotinib HCl 40 HPMCP HP55 — 156 912 1711 — 100/500 mg 150/750 mg 30 & 48 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 33 64 301 — 500 mg 750 mg 715 mg 56 & 57 I/P + S Nilotinib HCl 40 HPMCP HP55 Soluplus 48 65 81 100 (raw) 75 mg 112.5 mg 715 mg 37 & 52 I/P/S Nilotinib HCl 40 HPMCP HP55 Soluplus 163 516 663 — 500 mg 750 mg 715 mg

Conclusions Example 1

Experiments 17-23 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib HCl and a polymeric stabilizing and matrix-forming component. Particular improvements are achieved with the polymeric stabilizing and matrix-forming components hydroxypropyl methylcellulose phthalate (HPMCP HP55) and polyvinyl acetate phthalate (PVAP). These improvements are not obtained when physically mixing nilotinib HCl with a polymeric stabilizing and matrix-forming component. Experiments 24-36 clearly shows that a further solubility increase is obtained with hybrid nanoparticles produced by the methods of the invention with nilotinib HCl and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added. Particular improvements are achieved by the addition of a separate solubilizer such as polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus) or d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS). These improvements were not obtained when physically mixing nilotinib HCl, solubilizer and/or polymeric stabilizing and matrix-forming component (I+S or I+P+S). No particular improvements were obtained with hybrid nanoparticles with nilotinib HCl, a polymeric stabilizing and matrix-forming component and a solubilizer (I/P/S).

The results carried out in FaSSIF and FeSSIF, respectively, indicate that the stable, amorphous hybrid nanoparticles produced by the methods of the invention provide a similar increase in solubility. One issue with PKI formulation is the food effect. Several of the PKIs are labeled for administration in fasted state despite the fact that food in most cases increases their bioavailability. Low bioavailability might partly explain the digestive problems that are associated with the PKIs. The similar dissolution rate in FaSSIF and FeSSIF indicates that the hybrid nanoparticles produced by the methods of the invention (e.g. experiments 56/57) may reduce food effect and patient digestive problems by its solubility improvement that allows reducing dosage. Thus hybrid nanoparticles produced by the methods of the invention may be given in conjunction with food intake.

Example 2 Stable, Amorphous Hybrid Nanoparticles with Erlotinib HCl—Solubility at pH 6.5 and pH 5

A number of experiments were carried out, wherein erlotinib HCl represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 5, 30 and 90 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Further, experiments were carried out in an alternative solution at about pH 5, namely FeSSIF (Fed State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 7 and 8, where Table 7 provides data of concentration of erlotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution, whereas Table 8 provides data of % solubilized erlotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 90 minutes dissolution and the AUC increase of hybrid nanoparticles produced by the methods of the invention, compared to erlotinib HCl in raw, crystalline form added to the solution (experiments 58-68). In Tables 9 and 10, there is provided dissolution data in FeSSIF solution, presented similarly as Table 7 and 8 (experiments 69-73). In Table 11, data from a comparative experiment with similar hybrid nanoparticles produced by the methods of the invention, carried out in FaSSIF and FeSSIF, respectively (experiments 74-83). Table 12 presents further comparative data for experiments carried out in FaSSIF and FeSSIF, respectively, with stable, amorphous hybrid nanoparticles produced by the methods of the invention.

TABLE 7 Erlotinib - concentration of erlotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 58 I Erlotinib HCl 100 — — 28.9 6.25 4.6 (raw) 1000 mg 59 I + P Erlotinib HCl 100 HPMC-AS — 23 53.2 84 (raw) 1000 mg 2000 mg 60 I + S Erlotinib HCl 100 — Soluplus 92.8 156.6 176 (raw) 1000 mg 715 mg 61 I + S Erlotinib HCl 100 — TPGS 51.4 14.7 11.6 (raw) 1000 mg 1000 mg 62 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 96.7 256.6 361.8 (raw) 1000 mg 1850 mg 715 mg 63 I + P + S Erlotinib HCl 100 HPMC-AS TPGS 81.3 188.1 256.6 (raw) 1000 mg 1850 mg 1000 mg 64 I/P Erlotinib HCl 35 HPMC-AS — 83.4 79.6 44.8 1000 mg 1850 mg 65 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 187.3 269.7 284 1000 mg 1850 mg 715 mg 66 I/P + S Erlotinib HCl 35 HPMC-AS TPGS 155.2 210.6 225.3 1000 mg 1850 mg 1000 mg 67 I/P/S Erlotinib HCl 28 HPMC-AS Soluplus 90.1 95 96.4 1000 mg 1850 mg 715 mg 68 I/P/S Erlotinib HCl 26 HPMC-AS TPGS 93.7 85.4 52.8 1000 mg 1850 mg 1000 mg

TABLE 8 Percentage solubilized erlotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase of stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to erlotinib in raw, crystalline form added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 58 I Erlotinib HCl 100 — — 0.6 837 — (raw) 1000 mg 59 I + P Erlotinib HCl 100 HPMC-AS — 5.3 5126 6.1 (raw) 1000 mg 2000 mg 60 I + S Erlotinib HCl 100 — Soluplus 15.7 13328 15.9 (raw) 1000 mg 715 mg 61 I + S Erlotinib HCl 100 — TPGS 1.5 1744 2.1 (raw) 1000 mg 1000 mg 62 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 25.7 23210 27.7 (raw) 1000 mg 1850 mg 715 mg 63 I + P + S Erlotinib HCl 100 HPMC-AS TPGS 18.8 16912 20.2 (raw) 1000 mg 1850 mg 1000 mg 64 I/P Erlotinib HCl 35 HPMC-AS — 8.0 5978 7.1 1000 mg 1850 mg 65 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 27.0 22792 27.2 1000 mg 1850 mg 715 mg 66 I/P + S Erlotinib HCl 35 HPMC-AS TPGS 21.1 18038 21.5 1000 mg 1850 mg 1000 mg 67 I/P/S Erlotinib HCl 28 HPMC-AS Soluplus 9.5 8281 9.9 1000 mg 1850 mg 715 mg 68 I/P/S Erlotinib HCl 26 HPMC-AS TPGS 8.5 6619 7.9 1000 mg 1850 mg 1000 mg

TABLE 9 Erlotinib - concentration of erlotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 69 I Erlotinib HCl 100 — — 156.8 189.9 196 (raw) 1000 mg 70 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 25.5 75.1 126.2 (raw) 1000 mg 1850 mg 715 mg 71 I/P Erlotinib HCl 35 HPMC-AS — 258.2 402.1 464.5 1000 mg 1850 mg 72 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 260.1 422.8 498.8 1000 mg 1850 mg 715 mg 73 I/P/S Erlotinib HCl 28 HPMC-AS Soluplus 293.6 395.2 434.9 1000 mg 1850 mg 715 mg

TABLE 10 Percentage solubilized erlotinib HCl after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase of stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to erlotinib in raw, crystalline form added to the FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 69 I Erlotinib HCl 100 — — 19.0 16303 — (raw) 1000 mg 70 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 7.5 7360 0.5 (raw) 1000 mg 1850 mg 715 mg 71 I/P Erlotinib HCl 35 HPMC-AS — 40.2 34897 2.1 1000 mg 1850 mg 72 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 42.3 36835 2.3 1000 mg 1850 mg 715 mg 73 I/P/S Erlotinib HCl 28 HPMC-AS Soluplus 35.5 34244 2.1 1000 mg 1850 mg 715 mg

TABLE 11 Erlotinib - concentration of erlotinib HCl after 5, 30 and 90 minutes dissolution in FaSSIF and FeSSIF solution, respectively. Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 74 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 134.1 369.8 533.4 FaS (raw) 1000 mg 1850 mg 1430 mg SIF 75 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 24.4 88.8 154.4 FeS (raw) 1000 mg 1850 mg 1430 mg SIF 76 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 275.4 441.4 508 FaS 1000 mg 1850 mg 1430 mg SIF 77 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 292.2 476.2 546.5 FeS 1000 mg 1850 mg 1430 mg SIF 78 I/P/S Erlotinib HCl 23 HPMC-AS Soluplus 90.4 108 114.8 FaS 1000 mg 1850 mg 1430 mg SIF 79 I/P/S Erlotinib HCl 23 HPMC-AS Soluplus 259.3 354.8 405.5 FeS 1000 mg 1850 mg 1430 mg SIF 80 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 78.6 216.4 304.6 FaS (raw) 500 mg 925 mg 715 mg SIF 81 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 16.2 55.8 104.7 FeS (raw) 500 mg 925 mg 715 mg SIF 82 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 171.6 284.6 334.6 FaS 500 mg 925 mg 715 mg SIF 83 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 168.3 268.7 317.9 FeS 500 mg 925 mg 715 mg SIF

TABLE 12 Erlotinib - concentration of erlotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF and FeSSIF solution, respectively presented as comparative data. Drug Polymeric load stab. matrix. Compare Compare Compare ratio Component Solubilizer (%) (%) (%) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 58 & 69 I Erlotinib HCl 100 — — 543 3038 4261 (raw) 1000 mg 74 & 75 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 18 24 29 (raw) 1000 mg 1850 mg 1430 mg 80 & 81 I + P + S Erlotinib HCl 100 HPMC-AS Soluplus 21 26 34 (raw) 925 mg 715 mg 500 mg 64 & 71 I/P Erlotinib HCl 35 HPMC-AS — 310 505 1037 1000 mg 1850 mg 76 & 77 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 106 108 108 1000 mg 1850 mg 1430 mg 82 & 83 I/P + S Erlotinib HCl 35 HPMC-AS Soluplus 98 94 95 500 mg 925 mg 715 mg 78 & 79 I/P/S Erlotinib HCl 23 HPMC-AS Soluplus 287 329 353 1000 mg 1850 mg 1430 mg

Conclusions Example 2

The experiments show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention with erlotinib HCl and a polymeric stabilizing and matrix-forming component. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component hydroxypropyl methylcellulose acetate succinate (HPMC-AS). Experiments 65-66 and 72 show that a further solubility increase is obtained with hybrid nanoparticles produced by the methods of the invention with erlotinib HCl and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added. Particular improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus) and d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS). This improvement was not observed when the solubilizer was incorporated into the hybrid nanoparticles produced by the methods of the invention.

Physical mixes of erlotinib HCl with a solubilizer and/or HPMC AS improve also the solubility in FaSSIF (experiments 59, 60-61, 62-63) but not in FeSSIF (experiment 69-72). One issue with PKI formulation is the food effect. Several of the PKIs are labeled for administration in fasted state despite the fact that food in most cases increases their bioavailability. Low bioavailability might partly explain the digestive problems that are associated with the PKIs. The data indicates that the hybrid nanoparticles produced by the methods of the invention may reduce food effect and patient digestive problems by its equal solubility improvement in both FaSSIF and FeSSIF (experiment 76/77 and 82/83) that moreover potentially may allow reducing of dosage. Thus stable, amorphous hybrid nanoparticles produced by the methods of the invention may be given in conjunction with food intake.

Example 3 Stable, Amorphous Hybrid Nanoparticles with Pazopanib—Solubility at pH 6.5 and pH 5

A number of experiments were carried out, wherein pazopanib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 5, 30 and 90 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Further, experiments were carried out in an alternative solution at about pH 5, namely FeSSIF (Fed State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 13 and 14, where Table 13 provides data of concentration of pazopanib (mg/L) after 5, 30 and 90 minutes dissolution, whereas Table 14 provides data of % solubilized pazopanib after 30 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 90 minutes dissolution and the AUC increase with hybrid nanoparticles produced by the methods of the invention, compared to pazopanib in raw, crystalline form added to the solution (experiments 84-93). In Tables 15 and 16, there is provided dissolution data in FeSSIF solution, presented similarly as Table 13 and 14 (experiments 94-101). In Table 17, data from a comparative experiment with similar hybrid nanoparticles produced by the methods of the invention, carried out in FaSSIF and FeSSIF, respectively (experiments 102-109). Table 18 presents further comparative data for experiments carried out in FaSSIF and FeSSIF, respectively, with hybrid nanoparticles produced by the methods of the invention.

TABLE 13 Pazopanib - concentration of pazopanib (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 84 I Pazopanib (raw) 100 — — 46.2 24.4 15.0 1000 mg 85 I + P Pazopanib (raw) 100 PVP 90K — 82.7 83.8 67.7 1000 mg 2000 mg 86 I + S Pazopanib (raw) 100 — Soluplus 116.3 177.7 204.3 1000 mg 357 mg 87 I + S Pazopanib (raw) 100 — Soluplus 177.6 270.8 324.2 1000 mg 715 mg 88 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 198.8 312.2 394.1 1000 mg 1857 mg 715 mg 89 I + P + S Pazopanib (raw) 100 PVP 90K TPGS 182.6 196.7 49.2 1000 mg 1857 mg 1000 mg 90 I/P Pazopanib 35 PVP 90K — 89.4 103.4 92.8 1000 mg 1857 mg 91 I/P + S Pazopanib 35 PVP 90K Soluplus 238.9 409.4 469.3 1000 mg 1857 mg 715 mg 92 I/P + S Pazopanib 35 PVP 90K TPGS 207.5 244.8 76.3 1000 mg 1857 mg 1000 mg 93 I/P/S Pazopanib 28 PVP 90K Soluplus 127.2 128.3 82.0 1000 mg 1857 mg 715 mg

TABLE 14 Percentage solubilized pazopanib after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to pazopanib in raw, crystalline form added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 84 I Pazopanib (raw) 100 — — 2.4 2180 — 1000 mg 85 I + P Pazopanib (raw) 100 PVP 90K — 8.4 6833 3.1 1000 mg 2000 mg 86 I + S Pazopanib (raw) 100 — Soluplus 17.8 15426 7.1 1000 mg 357 mg 87 I + S Pazopanib (raw) 100 — Soluplus 27.1 23899 11.0 1000 mg 715 mg 88 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 31.2 28074 12.9 1000 mg 1857 mg 715 mg 89 I + P + S Pazopanib (raw) 100 PVP 90K TPGS 19.7 12575 5.8 1000 mg 1857 mg 1000 mg 90 I/P Pazopanib 35 PVP 90K — 10.3 8520 3.9 1000 mg 1857 mg 91 I/P + S Pazopanib 35 PVP 90K Soluplus 40.9 35062 16.1 1000 mg 1857 mg 715 mg 92 I/P + S Pazopanib PVP 90K TPGS 24.5 15806 7.3 1000 mg 1857 mg 1000 mg 93 I/P/S Pazopanib 28 PVP 90K Soluplus 12.8 9821 4.5 1000 mg 1857 mg 715 mg

TABLE 15 Pazopanib - concentration of pazopanib (mg/L) after 5, 30 and 90 minutes dissolution in FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 94 I Pazopanib (raw) 100 — — 231.3 321.4 239.3 1000 mg 95 I + P Pazopanib (raw) 100 PVP 90K — 234.8 309.7 269.7 1000 mg 2000 mg 96 I + S Pazopanib (raw) 100 — Soluplus 209.3 309.6 229.1 1000 mg 357 mg 97 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 307.5 475.3 578.0 1000 mg 1857 mg 715 mg 98 I + P + S Pazopanib (raw) 100 PVP 90K TPGS 320.9 395.1 325.6 1000 mg 1857 mg 1000 mg 99 I/P Pazopanib 35 PVP 90K — 348.4 362.1 335.8 1000 mg 1857 mg 100 I/P + S Pazopanib 35 PVP 90K Soluplus 450.0 684.4 777.6 1000 mg 1857 mg 715 mg 101 I/P/S Pazopanib 28 PVP 90K Soluplus 226.1 347.3 361.0 1000 mg 1857 mg 715 mg

TABLE 16 Percentage solubilized pazopanib after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to pazopanib in raw, crystalline form added to the FeSSIF solution (pH 5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 94 I Pazopanib (raw) 100 — — 32.1 24308 — 1000 mg 95 I + P Pazopanib (raw) 100 PVP 90K — 31.0 24775 1.0 1000 mg 2000 mg 96 I + S Pazopanib (raw) 100 — Soluplus 31.0 23171 1.0 1000 mg 357 mg 97 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 47.5 42153 1.7 1000 mg 1857 mg 715 mg 98 I + P + S Pazopanib (raw) 100 PVP 90K TPGS 39.5 31373 1.3 1000 mg 1857 mg 1000 mg 99 I/P Pazopanib 35 PVP 90K — 36.2 30689 1.3 1000 mg 1857 mg 100 I/P + S Pazopanib 35 PVP 90K Soluplus 68.4 59165 2.4 1000 mg 1857 mg 715 mg 101 I/P/S Pazopanib 28 PVP 90K Soluplus 34.7 28982 1.2 1000 mg 1857 mg 715 mg

TABLE 17 Pazopanib - concentration of pazopanib after 5, 30 and 90 minutes dissolution in FaSSIF and FeSSIF solution, respectively. Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 102 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 76.8 113.8 139.6 FaSSIF 300 mg 557 mg 428 mg 103 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 116.7 193.6 246.9 FeSSIF 300 mg 557 mg 428 mg 104 I/P + S Pazopanib 35 PVP 90K Soluplus 154.7 214.7 223 FaSSIF 300 mg 557 mg 428 mg 105 I/P + S Pazopanib 35 PVP 90K Soluplus 186 273.3 303.1 FeSSIF 300 mg 557 mg 428 mg 106 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 261.1 421.6 508.5 FaSSIF 1000 mg 1857 mg 1428 mg 107 I + P + S Pazopanib (raw) 100 PVP 90K Soluplus 275.8 495.4 588.0 FeSSIF 1000 mg 1857 mg 1428 mg 108 I/P + S Pazopanib 35 PVP 90K Soluplus 508.9 705.8 758.4 FeSSIF 1000 mg 1857 mg 1428 mg 109 I/P + S Pazopanib 35 PVP 90K Soluplus 469.1 715.2 747.4 FeSSIF 1000 mg 1857 mg 1428 mg

TABLE 18 Pazopanib - concentration of pazopanib (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF and FeSSIF solution, respectively presented as comparative data. Drug Polymeric load stab. matrix. Compare Compare Compare ratio Component Solubilizer (%) (%) (%) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 84 & 94 I Pazopanib 100 — — 501 1317 1595 (raw) 1000 mg 85 & 95 I + P Pazopanib 100 PVP 90K — 284 370 398 (raw) 1857 mg 1000 mg 87 & 96 I + S Pazopanib 100 — Soluplus 118 114 71 (raw) 715 mg 1000 mg 88 & 97 I + P + S Pazopanib 100 PVP 90K Soluplus 155 152 147 (raw) 1857 mg 715 mg 1000 mg 102 & I + P + S Pazopanib 100 PVP 90K Soluplus 152 170 177 103 (raw) 557 mg 428 mg 300 mg 90 & 99 I/P Pazopanib 35 PVP 90K — 390 350 362 1000 mg 1857 mg 89 & I/P + S Pazopanib 35 PVP 90K Soluplus 188 167 166 100 1000 mg 1857 mg 715 mg 104 & I/P + S Pazopanib 35 PVP 90K Soluplus 120 127 136 105 300 mg 557 mg 428 mg 93 & I/P/S Pazopanib 28 PVP 90K Soluplus 178 271 440 101 1000 mg 1857 mg 715 mg

Conclusions Example 3

The experiments show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention with pazopanib and a polymeric stabilizing and matrix-forming component. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component polyvinylpyrrolidone K-90 (PVP 90K). Experiments 91-92 show that a further solubility increase is obtained with hybrid nanoparticles produced by the methods of the invention with pazopanib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added. Particular improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus) and d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS). This improvement was not observed when the solubilizer was incorporated into the hybrid nanoparticles produced by the methods of the invention.

The results carried out in FaSSIF and FeSSIF, respectively, indicates that the hybrid nanoparticles produced by the methods of the invention provide a similar increase in solubility. One issue with PKI formulation is the food effect. Several of the PKIs are labeled for administration in fasted state despite the fact that food in most cases increases their bioavailability. Low bioavailability might partly explain the digestive problems that are associated with the PKIs. The similar dissolution rate in FaSSIF and FeSSIF indicates that the hybrid nanoparticles produced by the methods of the invention may reduce food effect and patient digestive problems by its equal solubility improvement in both FaSSIF and FeSSIF (experiments 89/100 and 104/105) that moreover allows reducing dosage. Thus stable, amorphous hybrid nanoparticles produced by the methods of the invention may be given in conjunction with food intake.

Example 4 Stable, Amorphous Hybrid Nanoparticles with Lapatinib—Solubility at pH 6.5

A number of experiments were carried out, wherein lapatinib base or lapatinib ditosylate salt represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 5, 30 and 90 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 19 and 20, where Table 19 provides data of concentration of lapatinib (mg/L) after 5, 30 and 90 minutes dissolution, whereas Table 20 provides data of % solubilized lapatinib after 30 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 90 minutes dissolution and the AUC increase with hybrid nanoparticles produced by the methods of the invention, compared to non-formulated lapatinib ditosylate salt added to the solution (experiments 110-126).

TABLE 19 Lapatinib - concentration of lapatinib (mg/L) after 5, 30 and 90 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 110 I Lapatinib (base) 100 — — 2.9 6.0 6.5 2000 mg 111 I Lapatinib (salt) 100 — — 57.7 132.2 124.2 2000 mg 112 I + S Lapatinib (salt) 100 — Soluplus 67.6 142.9 140.0 2000 mg 285 mg 113 I-S Lapatinib (salt) 100 — Soluplus 144.7 283.6 204.0 2000 mg 645 mg 114 I + P Lapatinib (base) 100 HPC LF — 1.9 4.9 6.1 2000 mg 4000 mg 115 I + P Lapatinib (salt) 100 HPC LF — 56.7 93.8 81.8 2000 mg 4000 mg 116 I + P + S Lapatinib (base) 100 HPC LF Soluplus 5.5 22.5 52.0 660 mg 340 mg 715 mg 117 I + P + S Lapatinib (salt) 100 HPC LF Soluplus 71.7 182.5 240.4 660 mg 340 mg 715 mg 118 I + P + S Lapatinib (base) 100 HPC LF TPGS 11.8 40.6 82.9 660 mg 340 mg 1000 mg 119 I + P + S Lapatinib (salt) 100 HPC LF TPGS 65.1 176.7 175.3 660 mg 340 mg 1000 mg 120 I/P Lapatinib (base) 66 HPC EF — 162.5 184.0 157.1 660 mg 340 mg 121 I/P Lapatinib (base) 66 HPC LF — 190.9 193.5 48.0 660 mg 340 mg 122 I/P + S Lapatinib (base) 66 HPC EF Soluplus 220.4 259.6 280.0 660 mg 340 mg 715 mg 123 I/P + S Lapatinib (base) 66 HPC LF Soluplus 200.7 315.6 327.6 660 mg 340 mg 715 mg 124 I/P + S Lapatinib (base) 66 HPC EF TPGS 202.2 237.5 242.5 660 mg 340 mg 500 mg 125 I/P + S Lapatinib (base) 66 HPC LF TPGS 288.4 327.3 301.5 660 mg 340 mg 500 mg 126 I/P/S Lapatinib (base) 66 HPC LF Soluplus 57.6 107.2 126.3 660 mg 340 mg 715 mg

TABLE 20 Percentage solubilized lapatinib after 30 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 90 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated lapatinib ditosylate salt added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 90 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 30 min. Mg/min/L increase 110 I Lapatinib (base) 100 — — 0.3 494 — 2000 mg 111 I Lapatinib (salt) 100 — — 6.6 10210 — 2000 mg 112 I + S Lapatinib (salt) 100 — Soluplus 7.1 11287 1.1 2000 mg 285 mg 113 I + S Lapatinib (salt) 100 — Soluplus 14.2 20344 2.0 2000 mg 645 mg 114 I + P Lapatinib (base) 100 HPC LF — 0.2 420 0.04 2000 mg 4000 mg 115 I + P Lapatinib (salt) 100 HPC LF — 4.7 7291 0.7 2000 mg 4000 mg 116 I + P + S Lapatinib (base) 100 HPC LF Soluplus 3.4 2599 0.3 660 mg 340 mg 715 mg 117 I + P + S Lapatinib (salt) 100 HPC LF Soluplus 27.7 16044 1.6 660 mg 340 mg 715 mg 118 I + P + S Lapatinib (base) 100 HPC LF TPGS 6.2 4390 0.4 660 mg 340 mg 1000 mg 119 I + P + S Lapatinib (salt) 100 HPC LF TPGS 26.8 13745 1.3 660 mg 340 mg 1000 mg 120 I/P Lapatinib (base) 66 HPC EF — 27.9 14971 1.5 660 mg 340 mg 121 I/P Lapatinib (base) 66 HPC LF — 29.3 12527 1.2 660 mg 340 mg 122 I/P + S Lapatinib (base) 66 HPC EF Soluplus 39.3 22739 2.2 660 mg 340 mg 715 mg 123 I/P + S Lapatinib (base) 66 HPC LF Soluplus 47.8 26252 2.6 660 mg 340 mg 715 mg 124 I/P + S Lapatinib (base) 66 HPC EF TPGS 36.0 20402 2.0 660 mg 340 mg 500 mg 125 I/P + S Lapatinib (base) 66 HPC LF TPGS 49.6 27281 2.7 660 mg 340 mg 500 mg 126 I/P/S Lapatinib (base) 66 HPC LF Soluplus 16.2 9209 0.9 660 mg 340 mg 715 mg

Conclusions Example 4

The experiments 122-125 clearly shows that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with lapatinib, in particular lapatinib base and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component hydroxypropyl cellulose EF and hydroxypropyl cellulose LF. Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus) and d-α-tocopherol acid polyethylene glycol 1000 succinate (TPGS).

Example 5 Stable, Amorphous Hybrid Nanoparticles with Nilotinib HCl Solubility at pH 1.4

A number of experiments were carried out, wherein nilotinib HCl represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 5, 30 and 90 minutes dissolution in a solution at about pH 1.4, namely SGF (Simulated Gastric Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in SGF solution are provided below in Table 21, which provides percentage of solubilized nilotinib HCl from both a physical mix with nilotinib HCl in raw, crystalline form and hybrid nanoparticles produced by the methods of the invention after 5, 30 and 90 minutes dissolution. Nilotinib present in the physical mix of nilotinib HCl raw with the polymeric stabilizing and matrix-forming component PVAP and the solubilizer Soluplus (Exp. 129) is dissolved completely within 5 minutes in SGF while nilotinib is only partially dissolved after 90 min in SGF with hybrid nanoparticles produced by the methods of the invention, wherein the components are comprised as hybrid nanoparticles, with the addition of a solubilizer (Exp. 128) or without the addition of a solubilizer (Exp. 127).

TABLE 21 Nilotinib HCl - concentration of nilotinib HCl (mg/L) after 5, 30 and 90 minutes dissolution in SGF solution (pH 1.4). Drug Polymeric load stab. matrix. % % % ratio Component Solubilizer solubilized solubilized solubilized Exp Comp. Inhibitor (I) (%) (P) (S) 5 min 30 min 90 min 127 I/P Nilotinib HCl 100 PVAP — 32 40 42 500 mg 750 mg 128 I/P + S Nilotinib HCl 100 PVAP Soluplus 38 48 50 500 mg 750 mg 715 mg 129 I + P + S Nilotinib HCl 100 PVAP Soluplus 100 100 100 (raw) 750 mg 715 mg 1000 mg

Conclusions Example 5

The experiments 127-129 shows that a nilotinib HCl, in stable, amorphous hybrid nanoparticles produced by the methods of the invention (exp 127 and 128) are partially solubilized at pH 1.4. The stable, amorphous hybrid nanoparticles produced by the methods with a enteric coating polymer such as PVAP is partially protected from the acidic environment.

Example 6 Stable, Amorphous Hybrid Nanoparticles with Gefitinib—Solubility at pH 6.5

A number of experiments were carried out, wherein gefitinib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 22 and 23, where Table 22 provides data of concentration of gefitinib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 23 provides data of % solubilized gefitinib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase with hybrid nanoparticles produced by the methods of the invention, compared to non-formulated gefitinib added to the solution (experiments 131-137).

TABLE 22 Gefitinib - concentration of gefitinib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 131 I Gefitinib (raw) 100 — — 121.8 153.1 148.1 1000 mg 132 I + P + S Gefitinib (raw) 35 PVP30K Soluplus 63.6 158.3 191.1 1000 mg 1850 mg 715 mg 133 I + P + S Gefitinib (raw) 35 HPMCP HP55 Soluplus 70.6 230.3 296.4 1000 mg 1850 mg 715 mg 134 I/P Gefitinib 35 PVP30K — 501.2 267.2 250.9 1000 mg 1850 mg 135 I/P Gefitinib 35 HPMCP HP55 — 254.1 321.4 332.1 1000 mg 1850 mg 136 I/P + S Gefitinib 35 PVP30K Soluplus 561.4 430.2 410.9 1000 mg 1850 mg 715 mg 137 I/P + S Gefitinib 35 HPMCP HP55 Soluplus 319.8 576.3 594.2 1000 mg 1850 mg 715 mg

TABLE 23 Percentage solubilized gefitinib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated gefitinib added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min. AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 131 I Gefitinib (raw) 100 — — 15.3 5967 — 1000 mg 132 I + P + S Gefitinib (raw) 35 PVP30K Soluplus 15.8 6630 1.1 1000 mg 1850 mg 715 mg 133 I + P + S Gefitinib (raw) 35 HPMCP HP55 Soluplus 23.0 9826 1.6 1000 mg 1850 mg 715 mg 134 I/P Gefitinib 35 PVP30K — 26.7 10954 1.8 1000 mg 1850 mg 135 I/P Gefitinib 35 HPMCP HP55 — 32.1 12794 2.1 1000 mg 1850 mg 136 I/P + S Gefitinib 35 PVP30K Soluplus 43.0 12282 2.9 1000 mg 1850 mg 715 mg 137 I/P + S Gefitinib 35 HPMCP HP55 Soluplus 57.6 22774 3.8 1000 mg 1850 mg 715 mg

The experiments 131-137 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with gefitinib, in particular gefitinib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component polyvinylpyrrolidone K-30 (PVP 30K) and hydroxy propyl methyl cellulose phthalate (HPMCP HP55). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus).

Example 7 Stable, Amorphous Hybrid Nanoparticles with Dasatinib—Solubility at pH 6.5

A number of experiments were carried out, wherein dasatinib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 24 and 25, where Table 24 provides data of concentration of dasatinib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 25 provides data of % solubilized dasatinib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase with hybrid nanoparticles produced by the methods of the invention, compared to non-formulated dasatinib added to the solution (experiments 138-141).

TABLE 24 Dasatinib - concentration of dasatinib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 138 I Dasatinib (raw) 100 — — 34.5 59.7 63.5 1000 mg 139 I + P + S Dasatinib (raw) 35 Kollidon VA64 Soluplus 24.2 64.9 82.5 1000 mg 1850 mg 715 mg 140 I/P Dasatinib 35 Kollidon VA64 — 54.7 382.0 417.6 1000 mg 1850 mg 141 I/P + S Dasatinib 35 Kollidon VA64 Soluplus 199.9 599.8 643.8 1000 mg 1850 mg 715 mg

TABLE 25 Percentage solubilized dasatinib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated dasatinib added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min. AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 138 I Dasatinib (raw) 100 — — 6.0 2396 — 1000 mg 139 I + P + S Dasatinib (raw) 35 Kollidon VA64 Soluplus 6.5 2750 1.1 1000 mg 1850 mg 715 mg 140 I/P Dasatinib 35 Kollidon VA64 — 35.3 15252 6.4 1000 mg 1850 mg 141 I/P + S Dasatinib 35 Kollidon VA64 Soluplus 58.6 24156 10.1 1000 mg 1850 mg 715 mg

Experiments 138-141 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with dasatinib, in particular dasatinib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component copolyvidone (Kollidon VA64). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus).

Example 8 Stable, Amorphous Hybrid Nanoparticles with Sorafenib Tosylate—Solubility at pH 6.5

A number of experiments were carried out, wherein sorafenib tosylate represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 26 and 27, where Table 26 provides data of concentration of sorafenib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 27 provides data of % solubilized sorafenib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase of compositions, compared to non-formulated sorafenib tosylate added to the solution (experiments 142-145).

TABLE 26 Sorafenib tosylate - concentration of sorafenib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 142 I Sorafenib 100 — — 59.1 343.5 311.5 tosylate (raw) 1000 mg 143 I + P + S Sorafenib 35 HPMCP HP55 Soluplus 33.9 297.1 352.2 tosylate (raw) 1850 mg 715 mg 1000 mg 144 I/P Sorafenib 35 HPMCP HP55 — 245.3 520.3 613.8 tosylate 1850 mg 1000 mg 145 I/P + S Sorafenib 35 HPMCP HP55 Soluplus 335.1 1202.6 1738.1 tosylate 1850 mg 715 mg 2000 mg

TABLE 27 Percentage solubilized sorafenib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated sorafenib tosylate added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min. AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 142 I Sorafenib 100 — — 34.4 12001 — tosylate (raw) 1000 mg 143 I + P + S Sorafenib 35 HPMCP HP55 Soluplus 33.9 11588 1.0 tosylate (raw) 1850 mg 715 mg 1000 mg 144 I/P Sorafenib 35 HPMCP HP55 — 245.3 21838 1.8 tosylate 1850 mg 1000 mg 145 I/P + S Sorafenib 35 HPMCP HP55 Soluplus 335.1 52948 4.4 tosylate 1850 mg 715 mg 2000 mg

Experiments 138-141 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with dasatinib, in particular dasatinib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component hydroxy propyl methyl cellulose phthalate (HPMCP HP55). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus).

Example 9 Stable, Amorphous Hybrid Nanoparticles with Nilotinib Base—Solubility at pH 6.5

A number of experiments were carried out, wherein nilotinib base represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 28 and 29, where Table 28 provides data of concentration of nilotinib base (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 29 provides data of % solubilized nilotinib base after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase of compositions, compared to non-formulated nilotinib base added to the solution (experiments 146-149).

TABLE 28 Nilotinib base - concentration of nilotinib base (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 146 I/P Nilotinib base 40 HPMCP HP55 — 12.7 5.3 3.7 500 mg 750 mg 147 I/P Nilotinib base 40 PVAP — 12.3 8.6 7.0 500 mg 750 mg 148 I/P + S Nilotinib base 40 HPMCP HP55 Soluplus 136.8 88.8 41.2 500 mg 750 mg 715 mg 149 I/P + S Nilotinib base 40 PVAP Soluplus 20.7 115.9 60.4 500 mg 750 mg 715 mg

TABLE 29 Percentage solubilized nilotinib base after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non- formulated nilotinib base added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min. AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 146 I/P Nilotinib base 40 HPMCP HP55 — 1.1 242 8.3 500 mg 750 mg 147 I/P Nilotinib base 40 PVAP — 1.7 328 11.2 500 mg 750 mg 148 I/P + S Nilotinib base 40 HPMCP HP55 Soluplus 17.8 3529 120.9 500 mg 750 mg 715 mg 149 I/P + S Nilotinib base 40 PVAP Soluplus 23.2 3544 121.4 500 mg 750 mg 715 mg

Example 10 Stable, Amorphous Hybrid Nanoparticles with Crizotinib—Solubility at pH 6.5

A number of experiments were carried out, wherein crizotinib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above. Representative results in FaSSIF solution are provided below in Table 30 and 31, where Table 30 provides data of concentration of crizotinib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 31 provides data of % solubilized crizotinib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated crizotinib added to the solution (experiments 150-156).

TABLE 30 Crizotinib - concentration of crizotinib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 150 I Crizotinib 100 — — 89.3 226.5 295.6 (raw) 1000 mg 151 I + P + S Crizotinib 25 PVP30K Soluplus 176.2 368.5 414.6 (raw) 1000 mg 3000 mg 715 mg 152 I + P + S Crizotinib 25 PVP30K Cremophor 161.2 428.4 497.7 (raw) 1000 mg 3000 mg RH40 715 mg 153 I/P Crizotinib 25 PVP30K — 325.9 390.4 398.8 1000 mg 3000 mg 154 I/P Crizotinib 25 Kollidon VA64 — 297.5 447.6 449.9 1000 mg 3000 mg 155 I/P + S Crizotinib 25 PVP30K Soluplus 457.6 581.4 578.9 1000 mg 3000 mg 715 mg 156 I/P + S Crizotinib 25 PVP30K Cremophor 573.9 855.1 867.2 1000 mg 3000 mg RH40 715 mg

TABLE 31 Percentage solubilized crizotinib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated crizotinib added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 150 I Crizotinib 100 — — 22.7 16773 (raw) 1000 mg 151 I + P + S Crizotinib 25 PVP30K Soluplus 36.8 27185 1.6 (raw) 1000 mg 3000 mg 715 mg 152 I + P + S Crizotinib 25 PVP30K Cremophor 42.8 30423 1.8 (raw) 1000 mg 3000 mg RH40 715 mg 153 I/P Crizotinib 25 PVP30K — 39.1 29958 1.8 1000 mg 3000 mg 154 I/P Crizotinib 25 Kollidon VA64 — 44.8 33611 2.0 1000 mg 3000 mg 155 I/P + S Crizotinib 25 PVP30K Soluplus 58.1 44862 2.7 1000 mg 3000 mg 715 mg 156 I/P + S Crizotinib 25 PVP30K Cremophor 85.5 64338 3.8 1000 mg 3000 mg RH40 715 mg

Experiments 150-156 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with crizotinib, in particular crizotinib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component polyvinylpyrrolidone K-30 (PVP 30K) and copolyvidone (Kollidon VA64). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus) and PEG-40 hydrogenated castor oil (Cremophor RH40).

Example 11 Stable, Amorphous Hybrid Nanoparticles with Axitinib—Solubility at pH 6.5

A number of experiments were carried out, wherein axitinib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above. Representative results in FaSSIF solution are provided below in Table 32 and 33, where Table 32 provides data of concentration of axitinib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 33 provides data of % solubilized axitinib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated axitinib added to the solution (experiments 157-163).

TABLE 32 Axitinib - concentration of axitinib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 157 I Axitinib 100 — — 0.6 0.6 0.6 (raw) 500 mg 158 I + P + S Axitinib 25 Kollidon VA64 Soluplus 0.2 0.8 4.3 (raw) 500 mg 1500 mg 715 mg 159 I + P + S Axitinib 25 HPMC AS Soluplus 0.2 3.0 3.1 (raw) 500 mg 1500 mg 715 mg 160 I/P Axitinib 25 Kollidon VA64 — 71.1 25.9 9.1 500 mg 1500 mg 161 I/P Axitinib 25 HPMC AS — 17.6 21.0 16.4 500 mg 1500 mg 162 I/P + S Axitinib 25 Kollidon VA64 Soluplus 77.6 223.6 266.1 500 mg 1500 mg 715 mg 163 I/P + S Axitinib 25 HPMC AS Soluplus 40.3 110.3 129.9 500 mg 1500 mg 715 mg

TABLE 33 Percentage solubilized axitinib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated axitinib added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 157 I Axitinib 100 — — 0.1 47 (raw) 500 mg 158 I + P + S Axitinib 25 Kollidon VA64 Soluplus 0.2 126 2.7 (raw) 500 mg 1500 mg 715 mg 159 I + P + S Axitinib 25 HPMC AS Soluplus 0.6 193 4.1 (raw) 500 mg 1500 mg 715 mg 160 I/P Axitinib 25 Kollidon VA64 — 5.2 3255 69.0 500 mg 1500 mg 161 I/P Axitinib 25 HPMC AS — 4.2 1571 33.0 500 mg 1500 mg 162 I/P + S Axitinib 25 Kollidon VA64 Soluplus 44.7 16070 341.0 500 mg 1500 mg 715 mg 163 I/P + S Axitinib 25 HPMC AS Soluplus 22.1 7954 169.0 500 mg 1500 mg 715 mg

Experiments 157-163 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with axitinib, in particular axitinib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component copolyvidone (Kollidon VA64) and hydroxypropyl methylcellulose acetate succinate (HPMC AS). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus).

Example 12 Stable, Amorphous Hybrid Nanoparticles with Vemurafenib—Solubility at pH 6.5

A number of experiments were carried out, wherein vemurafenib represented the PKI. The experiments were carried out by measuring concentration of PKI (mg/L) after 3, 40 and 80 minutes dissolution in a solution at about pH 6.5, namely FaSSIF (Fasted State Simulated Intestinal Fluid). Samples of the solution were taken at various time intervals and the amount of PKI was measured by the dissolution measurement assay described above.

Representative results in FaSSIF solution are provided below in Table 34 and 35, where Table 34 provides data of concentration of vemurafenib (mg/L) after 3, 40 and 80 minutes dissolution, whereas Table 35 provides data of % solubilized vemurafenib after 40 minutes dissolution, the Area Under the Curve (AUC—mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated vemurafenib added to the solution (experiments 164-170).

TABLE 34 Vemurafenib - concentration of vemurafenib (mg/L) after 3, 40 and 80 minutes dissolution in FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. Conc Conc Conc ratio Component Solubilizer (mg/L) (mg/L) (mg/L) Exp Comp. Inhibitor (I) (%) (P) (S) 3 min 40 min 80 min 164 I Vemurafenib 100 — — 0.3 0.3 0.4 (raw) 500 mg 165 I + P + S Vemurafenib 25 Kollidon VA64 Soluplus 0.2 0.2 0.4 (raw) 500 mg 1500 mg 715 mg 166 I + P + S Vemurafenib 25 CAP Soluplus 0.1 0.2 0.4 (raw) 500 mg 1500 mg 715 mg 167 I/P Vemurafenib 25 Kollidon VA64 — 35.5 107.6 122.9 500 mg 1500 mg 168 I/P Vemurafenib 25 CAP — 75.1 47.5 11.8 500 mg 1500 mg 169 I/P + S Vemurafenib 25 Kollidon VA64 Soluplus 27.4 111.3 172.3 500 mg 1500 mg 715 mg 170 I/P + S Vemurafenib 25 CAP Soluplus 55.4 105.7 118.9 500 mg 1500 mg 715 mg

TABLE 35 Percentage solubilized vemurafenib after 40 minutes dissolution, the Area Under the Curve (AUC - mg/min/L) during 80 minutes dissolution and the AUC increase with stable, amorphous hybrid nanoparticles produced by the methods of the invention, compared to non-formulated vemurafenib added to the FaSSIF solution (pH 6.5). Drug Polymeric load stab. matrix. AUC/ ratio Component Solubilizer % solubilized 80 min AUC Exp Comp. Inhibitor (I) (%) (P) (S) 40 min. Mg/min/L increase 164 I Vemurafenib 100 — — 0.1 27 (raw) 500 mg 165 I + P + S Vemurafenib 25 Kollidon VA64 Soluplus 0.1 21 0.8 (raw) 500 mg 1500 mg 715 mg 166 I + P + S Vemurafenib 25 CAP Soluplus 0.0 18 0.7 (raw) 500 mg 1500 mg 715 mg 167 I/P Vemurafenib 25 Kollidon VA64 — 21.5 7669 288.0 500 mg 1500 mg 168 I/P Vemurafenib 25 CAP — 9.5 3761 141.0 500 mg 1500 mg 169 I/P + S Vemurafenib 25 Kollidon VA64 Soluplus 22.3 8564 322.0 500 mg 1500 mg 715 mg 170 I/P + S Vemurafenib 25 CAP Soluplus 21.1 7899 297.0 500 mg 1500 mg 715 mg

Experiments 164-170 show that a solubility increase is obtained with stable, amorphous hybrid nanoparticles produced by the methods of the invention, with vemurafenib, in particular vemurafenib and a polymeric stabilizing and matrix-forming component, wherein a separate solubilizer is added to the composition. Particular improvements are achieved with the polymeric stabilizing and matrix-forming component copolyvidone (Kollidon VA64) and cellulose acetate phthalate (CAP). Further, improvements are achieved by the addition of a separate solubilizer added, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (Soluplus).

Example 13 Dissolution Rate Measurement in Sink Conditions Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention

Dissolution measurement in sink conditions of stable, amorphous hybrid nanoparticles produced by the methods of the invention were measured in a method consisting of adding the wished amount of powder into a flow through cell system (SOTAX, Allschwill, Switzerland), mounting the cell onto its apparatus and then pumping the appropriate medium (typically FaSSIF, FeSSIF, SGF) through the powder. The temperature of the apparatus was set to 37° C. The amount of powder added into the cell depends on drug load of the powder: The exact amount of powder was calculated from results obtained from drug load analysis of the powders.

Typically, 3.5 to 7 mg PKI was added into the flow through cell and a flow rate between 8 and 16 ml medium/min (preferably about 8 ml medium/min) was pumped through the powder. One ml samples of the medium passing through the cell were collected at predetermined times. These samples were analyzed by HPLC (e.g. C18 column Eclipse, 4.6 mm×15 cm, 1 ml/min, detection 254-400 nm). Samples were taken after 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 and 40 min from the moment the medium comes out from the flow through cell. The accumulated % solubilized of the amount of active substance added into the flow through cell was calculated and plotted against time (min). The initial slope (“initial dissolution rate”) of the graph was estimated, as measured during 0 to 10 minutes, and taken as the dissolution rate of the material in sink condition at 37° C. in the given dissolution medium.

Each experiment comprises a comparison between the PKI in raw form with stable, amorphous hybrid nanoparticles produced by the methods of the invention with the inhibitor and a representative polymeric stabilizing and matrix-forming component.

Example 13.1 Dissolution Rate Measurement in Sink Conditions with Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention, Comprising Nilotinib HCl

In experiments with nilotinib HCl, 4 mg was weighed in the flow through cell (experiment 500) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib base and the polymeric stabilizing and matrix-forming component HPMCP HP55 (experiment 501). The results are depicted in Table 36 below.

TABLE 36 Nilotinib HCl sink condition in FaSSIF. Experiment 500 Experiment 501 Composition type I I/P Inhibitor (I) nilotinib HCl (raw) nilotinib base Polymeric stab. — HPMCP HP55 matrix. Component (P) Drug load % — 40% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.13 3.07 0.5 0.33 7.96 1 0.49 12.23 1.5 0.63 15.22 2 0.76 17.91 3 1.02 23.25 4 1.24 28.03 5 1.48 32.70 6 1.71 37.32 7 1.92 42.04 8 2.13 45.78 9 2.34 49.52 10 2.56 52.34 15 3.51 59.66 20 4.31 66.28 25 5.04 70.92 30 5.7 74.38 35 6.4 76.25 40 7.0 80.33 Initial Dissolution Rate EXP 500 0.27 EXP 501 6.58 Ratio 501/500 24.0

Experiments 500-501 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with nilotinib base, is superior to the initial dissolution rate of nilotinib HCl in raw, crystalline form.

Example 13.2 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Erlotinib HCl

In experiments with erlotinib HCl, 3.5 mg was weighed in the flow through cell (experiment 510) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with erlotinib HCl and the polymeric stabilizing and matrix-forming component HPMC AS (experiment 511). The results are depicted in Table 37 below.

TABLE 37 Erlotinib HCl sink condition in FaSSIF. Experiment 510 Experiment 511 Composition type I I/P Inhibitor (I) erlotinib HCl (raw) erlotinib HCl Polymeric stab. — HPMC AS matrix. Component (P) Drug load % — 35% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.26 2.3 0.5 0.49 3.9 1 0.63 5.4 1.5 0.71 6.4 2 0.77 7.2 3 0.85 8.5 4 0.91 9.5 5 0.96 10.3 6 1.01 11.1 7 1.06 11.8 8 1.10 12.5 9 1.13 13.1 10 1.17 13.8 20 1.58 19.3 30 1.93 22.0 40 2.24 24.6 Initial Dissolution Rate EXP 510 0.303 EXP 511 2.754 Ratio 511/510 9.1

Experiments 510-511 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with erlotinib HCl, is superior to the initial dissolution rate of erlotinib HCl in raw, crystalline form.

Example 13.3 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Pazopanib HCl

In experiments with pazopanib HCl, 3.5 mg was weighed in the flow through cell (experiment 520) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with pazopanib HCl and the polymeric stabilizing and matrix-forming component PVP90K (experiment 521). The results are depicted in Table 38 below.

TABLE 38 Pazopanib HCl sink condition in FaSSIF. Experiment 520 Experiment 521 Composition type I I/P Inhibitor (I) pazopanib pazopanib HCl HCl (raw) Polymeric stab. — PVP90K matrix. Component (P) Drug load % — 35% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 1.6 1.9 0.5 4.7 4.6 1 7.7 6.8 1.5 9.6 8.8 2 11.2 10.6 3 13.4 15.2 4 14.7 19.7 5 15.4 22.7 6 16.0 26.2 7 16.4 30.1 8 16.9 33.8 9 17.2 38.2 10 17.6 41.7 20 19.2 73.2 30 20.5 91.3 40 21.6 97.1 Initial Dissolution Dissolution Rate Rate (5-10 min) EXP 520 4.8 0.428 EXP 521 4.33 3.85 Ratio 521/520 0.9 9.0

Experiments 520-521 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with pazopanib HCl, is superior to the initial dissolution rate of pazopanib HCl in raw, crystalline form.

Example 13.4 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Lapatinib Ditosylate

In experiments with lapatinib ditosylate, 4 mg was weighed in the flow through cell (experiment 530) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with lapatinib base and the polymeric stabilizing and matrix-forming component HPC If (experiment 531). The results are depicted in Table 39 below.

TABLE 39 Lapatinib ditosylate sink condition in FaSSIF. Experiment 530 Experiment 531 Composition type I I/P Inhibitor (I) Lapatinib ditosylate (raw) Lapatinib base Polymeric stab. — HPC If matrix. Component (P) Drug load % — 66% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.032 0.442 0.5 0.088 1.736 1 0.141 3.053 1.5 0.190 4.448 2 0.238 5.771 3 0.332 7.504 4 0.422 8.783 5 0.505 9.736 6 0.582 10.573 7 0.655 11.209 8 0.725 11.732 9 0.790 12.179 10 0.851 12.576 20 1.272 14.128 30 1.607 15.168 40 1.944 15.802 Initial Dissolution Rate EXP 530 0.103 EXP 531 2.674 Ratio 531/530 25.9

Experiments 530-531 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with lapatinib base, is superior to the initial dissolution rate of lapatinib ditosylate in raw, crystalline form.

Example 13.5 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Gefitinib

In experiments with gefitinib, 3.5 mg was weighed in the flow through cell (experiment 540) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with gefitinib and the polymeric stabilizing and matrix-forming component HPMCP HP55 (experiment 541). The results are depicted in Table 40 below.

TABLE 40 Gefitinib sink condition in FaSSIF. Experiment 540 Experiment 541 Composition type I I/P Inhibitor (I) Gefitinib (raw) Gefitinib Polymeric stab. — HPMCP HP55 matrix. Component (P) Drug load % — 35% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.1 1.8 0.5 0.9 6.7 1 1.9 11.3 1.5 3.2 15.4 2 4.5 19.0 3 7.0 23.6 4 9.5 27.4 5 11.9 30.5 6 14.3 33.5 7 16.6 36.0 8 18.8 37.8 9 20.7 39.9 10 22.7 42.6 20 29.9 50.6 30 34.1 56.7 40 36.7 61.8 Initial Dissolution Rate EXP 540 2.2 EXP 541 8.6 Ratio 541/540 3.9

Experiments 540-541 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with gefitinib, is superior to the initial dissolution rate of the gefinib in raw, crystalline form.

Example 13.6 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Dasatinib

In experiments with dasatinib, 3.5 mg was weighed in the flow through cell (experiment 550) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with dasatinib and the polymeric stabilizing and matrix-forming component copolyvidon-Kollidon VA64 (experiment 551). The results are depicted in Table 41 below.

TABLE 41 Dasatinib sink condition in FaSSIF. Experiment 550 Experiment 551 Composition type I I/P Inhibitor (I) Dasatinib (raw) Dasatinib Polymeric stab. — Kollidon VA64 matrix. Component (P) Drug load % — 35% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.3 0.4 0.5 0.7 1.0 1 1.2 1.7 1.5 1.6 2.3 2 2.0 2.9 3 2.8 4.2 4 3.7 5.5 5 4.4 6.8 6 5.2 8.2 7 6.0 9.5 8 6.8 10.8 9 7.6 12.1 10 8.3 13.4 20 15.9 25.9 30 22.1 40.9 40 26.4 54.9 Initial Dissolution Rate EXP 550 0.8 EXP 551 1.3 Ratio 551/550 1.6

Experiments 550-551 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with dasatinib, is superior to the initial dissolution rate of the dasatinib raw, crystalline form.

Example 13.7 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Sorafenib Tosylate

In experiments with sorafenib tosylate, 3.5 mg was weighed in the flow through cell (experiment 560) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with sorafenib tosylate and the polymeric stabilizing and matrix-forming component HPMCP HP55 (experiment 561). The results are depicted in Table 42 below.

TABLE 42 Sorafenib tosylate sink condition in FaSSIF. Experiment 560 Experiment 561 Composition type I I/P Inhibitor (I) Sorafenib tosylate (raw) Sorafenib tosylate Polymeric stab. — HPMCP HP55 matrix. Component (P) Drug load % — 35% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 0.2 0.8 0.5 0.4 1.7 1 0.7 2.4 1.5 1.0 3.1 2 1.3 3.7 3 1.8 4.8 4 2.2 5.8 5 2.6 6.9 6 3.0 8.1 7 3.4 9.7 8 3.8 11.3 9 4.2 13.3 10 4.6 15.6 20 8.8 32.7 30 12.6 61.5 40 16.4 96.1 Initial Dissolution Rate EXP 560 0.47 EXP 561 1.17 Ratio 561/560 2.5

Experiments 560-561 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with sorafenib tosylate, is superior to the initial dissolution rate of sorafenib tosylate in raw, crystalline form.

Example 13.8 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Crizotinib

In experiments with crizotinib, 3.5 mg was weighed in the flow through cell (experiment 570) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with crizotinib and the polymeric stabilizing and matrix-forming component PVP 30K (experiment 571). The results are depicted in Table 43 below.

TABLE 43 Crizotinib sink condition in FaSSIF. Experiment 570 Experiment 571 Composition type I I/P Inhibitor (I) Crizotinib (raw) Crizotinib Polymeric stab. — PVP 30K matrix. Component (P) Drug load % — 25% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % 0 2.0 8.8 0.5 5.7 30.3 1 8.9 47.9 1.5 11.9 58.3 2 14.6 67.5 4 23.1 81.7 6 30.1 83.8 8 36.0 84.2 10 41.0 84.4 20 58.9 85.1 30 73.1 85.3 40 86.3 85.5 Initial Dissolution Rate EXP 570 6.6 EXP 571 33.3 Ratio 571/570 5.0

Experiments 570-571 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with crizotinib, is superior to the initial dissolution rate of crizotinib in raw, crystalline form.

Example 13.9 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Axitinib

In experiments with axitinib, 3.5 mg was weighed in the flow through cell (experiment 580) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with axitinib and the polymeric stabilizing and matrix-forming component Kollidon VA64 (experiment 581) or HPMC AS (experiment 582). The results are depicted in Table 44 below.

TABLE 44 Axitinib sink condition in FaSSIF. Experiment 580 Experiment 581 Experiment 582 Composition type I I/P I/P Inhibitor (I) Axitinib (raw) Axitinib Axitinib Polymeric stab. — Kollidon VA64 HPMC AS matrix. Component (P) Drug load % — 25% 25% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % % 0 0.03 0.75 0.22 0.5 0.06 1.60 0.59 1 0.08 2.33 1.04 1.5 0.11 2.97 1.50 2 0.13 3.56 1.92 4 0.23 6.03 3.25 6 0.31 7.76 4.39 8 0.40 9.74 5.34 10 0.49 11.81 6.17 20 0.97 22.04 9.03 30 1.46 27.42 11.43 40 1.96 30.53 13.52 Initial Dissolution Rate EXP 580 0.051 EXP 581 & 582 1.396 0.865 Ratio 581/580 27.5 17.1 & 582/580

Experiments 580-582 show that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with axitinib, is superior to the initial dissolution rate of axitinib in raw, crystalline form.

Example 13.10 Dissolution Rate Measurement in Sink Conditions of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention Comprising Vemurafenib

In experiments with vemurafenib, 3.5 mg was weighed in the flow through cell (experiment 590) and compared with stable, amorphous hybrid nanoparticles produced by the methods of the invention with vemurafenib and the polymeric stabilizing and matrix-forming component Kollidon VA64 (experiment 591) or CAP (experiment 592). The results are depicted in Table 45 below.

TABLE 45 Vemurafenib sink condition in FaSSIF. Experiment Experiment 590 Experiment 591 592 Composition type I I/P I/P Inhibitor (I) Vemurafenib (raw) Vemurafenib Vemurafenib Polymeric stab. — Kollidon VA64 CAP matrix. Component (P) Drug load % — 25% 25% Accumulated % of solubilized of remaining active substance at a given time (min) Min. % % % 0 0.0 0.1 0.4 0.5 0.0 0.2 1.1 1 0.0 0.3 1.8 1.5 0.0 0.4 2.4 2 0.0 0.5 3.1 4 0.0 1.2 6.3 6 0.0 1.9 9.4 8 0.0 2.4 11.0 10 0.0 2.9 12.1 20 0.0 4.7 14.9 30 0.1 5.9 16.8 40 0.1 7.1 18.3 Initial Dissolution Rate EXP 590 0.002 EXP 591 & 592 0.209 1.346 Ratio 591/590 104 673 & 592/590

Experiments 590-592 clearly shows that the initial dissolution rate of the stable, amorphous hybrid nanoparticles produced by the methods of the invention, with vemurafenib, is superior to the initial dissolution rate of vemurafenib in raw, crystalline form.

Example 14 In Vivo Measurement of Plasma Levels after Oral Administration of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention

Groups of four male beagle dogs received single oral doses (5 mg/kg) of capsule compositions comprising stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib base and either of the polymeric stabilizing and matrix-forming components PVAP or HPMCP HP55, optionally with addition of the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, and compared with a marketed formulation comprising nilotinib HCl. The stable, amorphous hybrid nanoparticles tested are as set out in experiments 146-149, in Example 9. The stomach contents of the dogs were either neutralized with a sodium bicarbonate solution 5 min prior to capsule dosing or acidified with an HCl-KCl buffer 10 min prior to dose. One group of dogs also received a single iv dose (1 mg/kg) of nilotinib. Plasma levels of nilotinib were determined with a selective LC-MS/MS method. There were no side-effects observed in any animal studied.

Results and Conclusions

Mean±SEM plasma concentration-time profiles of nilotinib base are shown in FIGS. 22-25, and pharmacokinetic parameters and results are displayed in Tables 46A and 46B.

Outliers were calculated and excluded based on if one value is a significant outlier from the rest at 95% confidence intervals (alpha=5%) according to Grubb's test. The critical Z value for the Grubb's test at the 95% confidence interval with n=4 is 1.48. Z=(Mean−Questionable value)/SD

TABLE 46A Pharmacokinetic data following single oral administration of different nilotinib compositions comprising stable, amorphous hybrid nanoparticles produced by the methods of the invention, in male dogs. I/P + S I/P Nilotinib I/P Marketed Marketed Nilotinib base/PVAP + Nilotinib nilotinib nilotinib base/PVAP Soluplus base/PVAP formulation formulation Exp 147 Exp 149 Exp 147 Acidic Neutral Acidic Acidic Neutral Stomach Stomach Stomach Stomach Stomach Cmax, 86 ± 52 73 ± 26 240 ± 87  360 ± 89  490 ± 350 ng/mL Tmax, hr 7.6 ± 11  1.3 ± 0.3 1.3 ± 0.3 1.3 ± 0.3 1.4 ± 0.5 T½, hr 9.9; 10.7 1.9 ± 0.3 4.3 ± 3.0 3.3 ± 2.0 3.4 ± 1.2 AUC 0-24 h, 400 ± 140 220 ± 90  650 ± 240 1260 ± 70  1820 ± 1200 ng * hr/mL F (%) 7.9 ± 2.9 4.4 ± 1.8 13 ± 5  25 ± 1  36 ± 24

Values are given as Mean±SD, except for T ½ of the Marketed nilotinib formulation given too acid stomach where only two values were obtained.

Intravenous (IV) data were obtained by constant rate IV infusion of 1 mg/kg, of a solution of Nilotinib at 0.2 mg/mL, in a 10% HPβCD, pH adjustment to pH 3.3 to 3.5. Co: 511±46 ng/mL; T½: 3.3±1.8 hr; AUC0-24 hr: 1000±300 ng*hr/mL.

TABLE 46B Pharmacokinetic data following single oral administration of different nilotinib formulations comprising stable, amorphous hybrid nanoparticles produced by the methods of the invention, in male dogs. I/P I/P I/P Nilotinib base/ I/P Nilotinib base/ Nilotinib base/ HPMCP HP55 + Nilotinib base/ HPMCP HP55 + HPMCP HP55 Soluplus HPMCP HP55 Soluplus Exp 146 Exp 148 Exp 146 Exp 148 Neutral Neutral Acidic Acidic Stomach Stomach Stomach Stomach Cmax, 210 ± 97  560 ± 220 380 ± 90  270 ± 130 ng/mL Tmax, hr 1.1 ± 0.5  1.3 ± 0.29 1.2 ± 0.3 1.0 ± 0.0 T½, hr 1.9 ± 0.2 3.0 ± 1.4 3.3 ± 1.3 3.8 ± 0.8 AUC 0-24 h, 730 ± 390 1600 ± 580  1230 ± 110  910 ± 630 ng * hr/mL F (%) 15 ± 8  32 ± 12 24 ± 2  18 ± 13

Values are given as Mean±SD

Intravenous (IV) data were obtained by constant rate IV infusion of 1 mg/kg, of a solution of Nilotinib at 0.2 mg/mL, in a 10% HPβCD, pH adjustment to pH 3.3 to 3.5. Co: 511±46 ng/mL; T½: 3.3±1.8 hr; AUC0-24 hr: 1000±300 ng*hr/mL.

The marketed nilotinib formulation administrated to an acidified stomach showed plasma levels about 2 times higher than those after the same formulation administered to a neutralized stomach. Both formulations comprising stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib base with PVAP and HPMCP HP55 as polymeric stabilizing and matrix-forming components showed significant improvements in plasma exposure, with plasma levels about 2-fold higher than those of the marketed formulation given to an acidified stomach. In addition, combining hybrid nanoparticles produced by the methods of the invention could give a plasma exposure that is be more or less independent of stomach pH.

Further improvements in oral availability were observed when formulations with stable, amorphous hybrid nanoparticles produced by the methods of the invention were combined with the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer. Thus, hybrid nanoparticles produced by the methods of the invention with nilotinib base with PVAP and HPMCP HP55 as polymeric stabilizing and matrix-forming components, where the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added and administered to an acidified stomach resulted in plasma levels 2.3- to 3.1-fold higher than those of the marketed formulation. In this study, high oral bioavailability was achieved with stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib base with HPMCP HP55 as polymeric stabilizing and matrix-forming components, where the solubilizer polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added (I/P+S) and administrated to neutralized stomach contents. In this case the exposure increased about 7-fold over that of the marketed oral formulation administered under the same neutralized conditions. Highest bioavailability, 36±24%, in this study was achieved when stable, amorphous hybrid nanoparticles produced by the methods of the invention with nilotinib base with PVAP as polymeric stabilizing and matrix-forming components was administered to a neutralized stomach. However, this study leg was also accompanied with the highest standard deviation in the study.

There was an improvement in the in vivo performance of the novel formulations of nilotinib with stable, amorphous hybrid nanoparticles produced by the methods of the invention, that are based on improving absorption and bioavailability by optimization of the solid state properties of the dosage form. The results of the in vivo study in dogs may predict similar absorption properties of hybrid nanoparticles produced by the methods of the invention, in patients, as there appears to be a close correlation in dog-human gastrointestinal drug absorption processes (Persson, E. M. et al. Pharm. Res. 2005, 22, 2141-2151). The hybrid nanoparticles produced by the methods of the invention, with advantageous absorption properties, also predicts that the oral doses used in clinical practice today may be lowered. Furthermore, the stable, amorphous hybrid nanoparticles produced by the methods of the invention may cause less pH-dependency in the absorption and bioavailability of PKIs.

Example 15 Measurement of Degree/Level of Stability of Stable, Amorphous Hybrid Nanoparticles Produced by the Methods of the Invention

In stability tests of stable, amorphous hybrid nanoparticles produced by the methods of the invention, it was shown that particles were stable over at least 11 months at room temperature (18-25° C.), as measured by X-Ray powder diffraction and dissolution rate by measurement of AUC.

In series of experiments with hybrid nanoparticles comprising nilotinib and HPMCP HP55 produced by the methods of the invention, the resulting particles provided stable, amorphous hybrid nanoparticles at 40% drug load (I/P nilotinib base/HPMCP HP55: exp 146), as measured by XRPD as well as dissolution rate by measurement of AUC. The material showed one glass transition temperature at ca 127° C., which indicate a single amorphous phase with inherent stability. Partially crystalline batches also processed similar inherent stability. 6 months storage at room temperature (18-25° C.) of partly crystalline hybrid nanoparticles at 40% drug load, I/P nilotinib base/HPMCP HP55, did not show any signs of physical instability.

Thermalgravimetric analysis provided a mass loss of 1.7% from ambient temperature to 120° C.

Dynamic vapor absorption analysis at 25° C. gave a relative mass increase of ca 7% from 0 to 90% RH (Three cycles from 0 to 90% RH, did not induce a phase change).

The high glass transition temperature, 1.7% mass loss from ambient temperature to 120° C. and moderate hygroscopicity propose an inherent stability. This is supported by stability testing of several batches at various conditions. The longest stability point is 12 months at room temperature (18-25° C.). No batches or conditions have shown any signs of physical instability (FIG. 27).

Modulated Differential Scanning Calorimetry (mDSC)

Modulated Differential Scanning calorimetry (mDSC) analysis was run on a TA Instruments Model Q200 (New Castle, USA), equipped with a RC90 refrigerated cooling system (Home Automation, New Orleans, USA). Samples were weighed to 7±2 mg in Tzero Low-mass aluminum pans and sealed with Tzero lids. They were then heated at a heating rate of 3° C./min from 0 to 170° C. with conventional modulation temperature amplitude of 1° C. and a modulation period of 40 seconds. Ultra-high purity nitrogen was used as purge gas at a flow rate of 50 mL/min. All data analyses were performed using TA Universal Analysis software, version 4.7A. Cell constant and temperature calibrations were conducted with the use of an indium standard prior to instrument operation. DSC results were evaluated in terms of both forward and reverse components of heat flow.

Thermogravimetry (TG) was performed on a Seiko TG/DTA 6200 and open 90 μl Pt-pans with ca 10 to 20 mg of sample and a nitrogen flow of 200 mL/min. The temperature program was ambient (20° C.) to 400° C. with a heating rate of 10° C./min. A blank was subtracted and the TG data was normalized with respect to sample size and analyzed using the Muse Standard Analysis software, version 6.1 U.

Dynamic Vapour Sorption (DVS)

The hygroscopicity of the samples was studied by Dynamic Vapor Sorption Gravimetry (DVS), using a DVS-1 (Surface Measurement Ltd., UK). Approximately 10 mg of the substance was weighed into a glass cup. The relative weight was recorded at 20 second interval when the target relative humidity (RH) over the sample was increased stepwise from 0% to 90%, and then similarly decreased back to 0% RH, with 10% RH per step. Each sample was run in three consecutive full cycles. The condition to proceed to the next level of RH was a weight change below or equal to 0.002% within 15 minutes, with a maximum total time per step of 24 hours. Due to slow equilibration in experiments of this type, the numbers obtained should be regarded as lower estimates of water uptake. The temperature was kept at 25° C.

X-Ray Powder Diffraction (XRPD)

XRD experiments were run on an X'Pert Pro diffractometer (PANanalytical, Almelo, Netherlands) set in Bragg-Brentano geometry. The diffractometer was equipped with 20 μm nickel filter and an X'Celerator RTMS detector with an active length of 2.122° 20. A representative sample was placed on a zero background quarts single crystal specimen support (Siltronix, Archamps, France). Experiments were run using Cu K_(α) radiation (45 kV and 40 mA) at ambient temperature and humidity. Scans were run in continuous mode in the range 4.5-40° 20 using automatic divergence and anti-scatter slits with observed length of 10 mm, a common counting time of 299.72 seconds, and step size of 0.0167° 2θ. Data collection was done using the application software X'Pert Data Collector V.2.2j and instrument control software V.2.1E, while pattern analysis was done using X'Pert Data Viewer V.1.2c (all software being from PANanalytical, Almelo, Netherlands).

Dissolution Rate by Measurement of AUC

The stable, amorphous hybrid nanoparticles described in Exp 171 & Exp172 (I/P) as set out below, were produced according to Exp 148, with nilotinib base, HPMCP HP55 and stored at room temperature for 11 months. The non-sink dissolution rate was tested at different time points and the results are presented in Table 47 and FIG. 26. Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer was added to enhance solubility. A comparison of the AUC over 80 minutes show clearly that the dissolution rate profile of the particles is practically unchanged after 11 months storage, e.g. the ratio between the AUC of particles produced and tested, compared to particles produced, tested and stored for 11 months is over 97%.

TABLE 47 Ratio 0 1 min 5 min 10 min 20 min 40 min 80 min AUC (%) Exp 171 (t = 0, 0 70.5 144.8 172.8 155.8 67.7 46.0 7411.9 n = 1) Exp 171 (t = 11 0 20.2 110.7 149.5 158.5 83.6 34.0 7234.5 97.6 months, n = 3) Standard 0 5.8 14.3 19.6 19.6 3.5 1.5 deviation 

1. A method of producing stable, amorphous hybrid nanoparticles comprising at least one protein kinase inhibitor and at least one polymeric stabilizing and matrix-forming component, comprising a) providing a first pressurized stream of said protein kinase inhibitor dissolved in a solvent; b) providing a second pressurized stream of antisolvent; wherein said at least one polymeric stabilizing and matrix-forming component is present in either said first or second stream; and c) mixing said first and second streams, and spraying the mixed stream at the outlet of a nozzle, whereby said hybrid nanoparticles are formed; followed by collecting said hybrid nanoparticles.
 2. The method of claim 1, wherein said polymeric stabilizing and matrix-forming component is present in said solvent.
 3. The method of claim 1, wherein said polymeric stabilizing and matrix-forming component is present in said antisolvent.
 4. The method according to claim 1, wherein at least one of said fluid streams is a supercritical fluid stream, preferably a super- or subcritical CO₂ fluid stream.
 5. The method according to claim 1, wherein said stream of antisolvent is a super- or subcritical CO₂ fluid stream.
 6. The method according to claim 1, wherein said stream of solvent is a super- or subcritical CO₂ fluid stream.
 7. The method according to claim 1, wherein said at least one polymeric stabilizing and matrix-forming component is present in a super- or subcritical CO₂ fluid stream.
 8. The method according to claim 1, wherein said first stream or said second stream is comprised of a super- or subcritical CO₂ fluid stream is provided at a temperature of about 25° C. or lower, at a pressure of from about 100 to about 150 bar.
 9. The method according to claim 1, wherein said at least one polymeric stabilizing and matrix-forming component is selected from methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose phthalate, polyvinylpyrrolidone, polyvinyl acetate phthalate, copolyvidone, crospovidon, methacrylic acid and ethylacrylate copolymer, methacrylate acid and methyl methacrylate copolymer, polyethylene glycol, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, DL lactide/glycolide copolymer, poly DL-lactide, cellulose acetate phthalate, carbomer homopolymer Type A, carbomer homopolymer Type B, aminoalkyl methacrylate copolymers and polaxamers.
 10. The method according to claim 1, wherein said at least one polymeric stabilizing and matrix-forming component is selected from hydroxypropyl methylcellulose phthalate, hydroxypropyl cellulose, copolyvidone, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate and polyvinylpyrrolidone.
 11. The method according to claim 1, wherein a solubilizer is added to the hybrid nanoparticles obtained in step c.
 12. The method of claim 11, wherein said solubilizer is selected from polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate and a hydrogenated castor oil.
 13. The method according to claim 1, wherein said hybrid nanoparticles comprising said protein kinase inhibitor provide an increased dissolution rate, compared to the dissolution rate of said protein kinase inhibitor in raw, crystalline form.
 14. The method of claim 13, wherein said dissolution rate is measured by a flow through cell system.
 15. The method of claim 13, claim 13, wherein said dissolution rate is measured within the initial 0 to 10 minutes of dissolution.
 16. The method according to claim 13, wherein said increased dissolution rate is measured in a solution as a dissolution rate ratio of said hybrid nanoparticles comprising said protein kinase inhibitor and said protein kinase inhibitor in raw, crystalline form.
 17. The method of claim 16, wherein said ratio is from about 1.5:1 to about 500:1.
 18. The method according to claim 13, wherein said dissolution rate is measured in a solution with a gastric pH.
 19. The method according to claim 13, wherein said dissolution rate is measured in a solution with an intestinal pH.
 20. The method according to claim 1, which produces hybrid nanoparticles that provide a solubility increase of said protein kinase inhibitor in a solution, said increase measured as the area under the curve (AUC) during about from 40 minutes to about 90 minutes, in said solution as compared with the AUC of said protein kinase inhibitor in raw, crystalline form.
 21. The method of claim 20, wherein said increase is from about 2:1 to about 10 000:1, wherein 1 represents the AUC of said protein kinase inhibitor in raw, crystalline form.
 22. The method of claim 20, wherein said increase is measured in a solution with gastric pH.
 23. The method according to claim 20, wherein said increase is measured in a solution with an intestinal pH.
 24. The method according to claim 1, wherein said amorphous hybrid nanoparticles are characterized by providing an amorphous powder X-ray diffraction pattern.
 25. The method according to claim 1, wherein the dissolution rate of said stable, amorphous hybrid nanoparticles remain stable to at least about 90%, after 9 months of storage or more, at room temperature.
 26. The method according to claim 1, wherein said protein kinase inhibitor is a tyrosine kinase inhibitor selected from the group consisting of lapatinib, pazopanib, nilotinib, erlotinib, dasatinib, gefitinib, sorafenib, crizotinib, axitinib, vemurafenib salts thereof, hydrates thereof, solvates thereof, and combinations thereof.
 27. The method according to claim 1, wherein said hybrid nanoparticles have an average particle diameter size of less than about 1000 nm.
 28. The method according to claim 1, wherein said hybrid nanoparticles have an average diameter size is less than about 500 nm.
 29. The method according to claim 1, wherein said solvent is an organic solvent selected from DMSO and trifluoroethanol, or a mixture thereof.
 30. The method according to claim 1, wherein said hybrid nanoparticles further comprise a solubilizer.
 31. The method of claim 30, wherein said solubilizer is polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer, d-α-tocopherol acid polyethylene glycol 1000 succinate or a hydrogenated castor oil.
 32. (canceled)
 33. The method according to claim 1, further comprising formulating said hybrid nanoparticles obtained from step (c) as a pharmaceutical composition, wherein said pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient. 